US intelligence agents believes the voice on a tape purportedly from Osama Bin Laden was authentic, the CIA and White House have said.
The message, in audio only, appeared on Wednesday on an Islamist website.
It threatened the EU over the re-printing of a cartoon satirising the Prophet Muhammad.
"It can be said with a high degree of confidence that it is in fact the voice of Osama Bin Laden," a CIA official told Reuters news agency.
The voice on the recording says the cartoon, re-published recently in all major Danish newspapers, was part of a "new crusade" in which Pope Benedict XVI had played a "large and lengthy role".
The Vatican dismissed the claims on Thursday as "absolutely baseless".
A spokesman also pointed out that the Pope and his advisers had "denounced the satirical campaign against Islam on more than one occasion".
The drawing, first published in 2005, depicts the Prophet Muhammad with a bomb in his turban.
The unnamed CIA official said the message was "well worn" and part of al-Qaeda's "ongoing propaganda campaign".
The White House said the tape appeared to show Bin Laden was trying "to establish himself as relevant and posture himself as the defender of Muslims".
The message came on the fifth anniversary of the US-led invasion of Iraq.
But the BBC's Jonathan Beale in Washington says that the message was probably not released to mark that anniversary, but rather the anniversary of the birth of the Prophet Muhammad, which Sunni Muslims mark on Thursday.
The Islamist website it appeared on has carried al-Qaeda messages in the past.
Over the audio is a graphic with a still image of Bin Laden holding an AK-47 and bearing the logo of al-Sahab, the media wing of al-Qaeda. There is a written translation of the message in English.
Last month, Denmark's leading newspapers reprinted one of 12 cartoons that first angered many Muslims when they were originally published in September 2005.
Anger in the Muslim world peaked in 2006 as newspapers in other countries published the cartoons.
Some of the protests turned violent and led to the torching of Danish diplomatic offices in Damascus and Beirut and dozens of deaths in Nigeria, Libya and Pakistan.
The Danish newspapers decided to republish the most controversial drawing after Danish intelligence said it had uncovered a plot to kill the cartoonist.
The message attributed to Bin Laden says attacks by Europeans on women and children "paled [in comparison] when you went overboard in your unbelief and... went to the extent of publishing these insulting drawings.
"This is the greatest misfortune and the most dangerous," the voice says.
"If there is no check on the freedom of your words, then let your hearts be open to the freedom of our actions".
It is not clear when the message was recorded. The last audio message attributed to Bin Laden appeared in November but he has not been seen on video since October 2004.
He is believed to be in hiding in the rugged border area between Pakistan and Afghanistan.