The Croatian navy has intercepted an Italian fishing boat, two days after Zagreb imposed a protection zone in its territorial waters.
A government official said the trawler was "illegally present" and it was escorted to a port on the island of Vis for investigation.
Police said the boat was in Croatian waters but not in the designated zone.
But the incident will raise tensions after the EU warned Zagreb the zone could harm its bid to become a member.
Overfishing
Croatia argues its fish stocks have been depleted by Italian overfishing.
It says that the government loses 300m euros (£219m) each year and complains that Italy's annual catch of 200,000 tonnes is ten times the size of its own.
The protected area covers 57,000sq km (22,800sq miles) of territorial waters.
Neighbouring Slovenia, which took over the EU presidency on 1 January, has strongly criticised the step.
It protested that Slovenia had supported Croatia's efforts to join the EU and as a result was "entitled to expect Croatia to act in a credible manner in this case".
A police spokeswoman, Zeljka Radosevic, told state radio that the boat had been spotted fishing illegally near a remote Croatian island.
"Crew members and the commander will be taken before a judge," Ms Radosevic said.
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