Iran's decision to build a secret facility represented a "direct challenge to the basic compact" of the global non-proliferation regime, US President Barack Obama said, making a statement in Pittsburgh, where he is hosting a G20 summit.
Despite Iran's assertions that the facility was for peaceful purposes, the new plant was "not consistent" with that goal, the US president said.
'Line in the sand'
Speaking alongside UK Prime Minister Gordon Brown and French President Nicolas Sarkozy, Mr Obama said it was time for Iran to begin meeting its international commitments.
BBC World Affairs correspondent Paul Reynolds
Iranian ambitions for this site are not known. It could be that they wanted a back-up in case their main plant at Natanz was attacked. But another fear is that they intended to enrich uranium more highly at the secret plant, to a level suitable for a nuclear explosion.
The discovery will strengthen the demands by the US and its allies for further sanctions to be imposed on Iran unless it suspends all enrichment, as required by the Security Council.
"Iran must comply with UN Security Council resolutions and make clear it is prepared to meet its responsibilities as a member of the community of nations," Mr Obama said.
Tehran would be held accountable for any failure to meet these responsibilities, he said.
Speaking after Mr Obama, the French and British leaders used strong language to insist that Iran would now have to disclose full details of its entire nuclear programme or face new and tougher sanctions.
Gordon Brown stressed that the US, France and UK were "at one" on the issue, and accused the Iranians of "serial deception".
There was now "no choice but to draw a line in the sand" over the nuclear issue, he said.
"Iran must abandon any military ambitions for its nuclear programme."
Mr Sarkozy said the situation was a challenge to the entire international community.
"Everything must be put on the table," the French president said, adding that the world needed to see a "step change" from Iran in the coming months.
Russian President Dmitry Medvedev, informed of the plant's existence by Mr Obama this week, said the second plant was against the requirements of UN Security Council resolutions.
Iran must co-operate fully with the UN watchdog, the International Atomic Energy Agency (IAEA), Mr Medvedev said.
China also said Iran should work with the UN watchdog, a foreign ministry spokesman said in Pittsburgh.
Speaking in New York, where he has been attending the UN General Assembly, Iran's President Ahmadinejad struck a defiant tone after the Pittsburgh proclamations.
"It's not a secret site. If it was, why would we have informed the IAEA about it a year ahead of time?" he said.
Ahmadinejad: 'We work within the framework of the IAEA'
He insisted Iran was acting within the boundaries of IAEA rules, saying it does not need to inform the agency of any new site until 180 days before any nuclear material is placed within it.
The existence of Iran's first enrichment plant, at Natanz, was only confirmed after intelligence emerged from Iranian exile groups several years ago.
Western governments are said to have known of the existence of the new enrichment plant for some time. Mr Obama was first told about it during the "transition" period before he took office in January, officials say.
In Washington, US officials said the Western nations decided to reveal their intelligence assessments when the Iranians realised the plant's secrecy was compromised.
IRAN'S NUCLEAR SITES
Iran insists that all its nuclear facilities are for energy, not military purposes
Bushehr: Nuclear power plant
Isfahan: Uranium conversion plant
Natanz: Uranium enrichment plant, 4,592 working centrifuges, with 3,716 more installed
Second enrichment plant: Existence revealed to IAEA in Sept 2009. Separate reports say it is near Qom, and not yet operational
The new facility is said to be underground at a mountain on the site of a former missile site belonging to the Iranian Revolutionary Guards, north-east of the holy city of Qom.
Construction on the facility - believed by the US to be large enough to contain 3,000 centrifuges, not large enough for commercial work - started in earnest in mid-2006, diplomatic sources said.
Iran's letter to the UN watchdog, the IAEA, on Monday informed it that "a new pilot fuel enrichment plant is under construction".
Iran told the agency that no nuclear material had been introduced into the plant, and enrichment levels would only be high enough to make nuclear fuel, not a bomb.
In response, the IAEA has requested Iran to "provide specific information and access to the facility as soon as possible", an IAEA statement adds.
The disclosure of the new plant comes one day after world leaders stressed the need for greater co-operation against nuclear proliferation and shortly before Iran is due to resume talks with international powers on the issue.
Since taking office in January, Mr Obama has told Tehran than he is ready for direct talks on the nuclear issue, but has had no firm response from Iran.
Earlier this month, Tehran agreed to "comprehensive" talks on a range of security issues - but made no mention of its own nuclear programme.
The talks are due to be held in Geneva on 1 October with Tehran and the five permanent UN Security Council members - US, UK, Russia, China and France - plus Germany.
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