The commission president's robust warning about Scotland's future status alarmed the devolved government in Edinburgh, forcing Nicola Sturgeon, the Scottish deputy first minister, to call for early talks about Scotland's legal rights should it gain independence.
In an interview with BBC News, Barroso said it was clear under commission rulings dating back to 2004 that a newly independent state which had emerged from an existing member state would be treated as a new applicant. That country would need to reach agreement on membership with all other 27 member states in order to join, he said, going into far greater detail than in his first, more cautious remarks on the issue in September.
"For European Union purposes, from a legal point of view, it is certainly a new state. If a country becomes independent it is a new state and has to negotiate with the EU," Barroso said.
"Now what I said, and it's our doctrine and it's clearly since 2004 in legal terms, if one part of a country – I'm not referring now to any specific one – wants to become an independent state, of course as an independent state it has to apply to the European Union for membership according to the rules. That's obvious."
Crucially for the UK government, Barroso said that the rest of the UK would, in principle, remain part of the EU without any need to rejoin or renegotiate the terms of its membership because of the "principle of continuity of the state".
Alex Salmond, the first minister, has repeatedly suggested that Scotland's independence would give the rest of the UK the same legal status as Scotland, as a successor state – a view the UK government's legal advisers have dismissed as highly unlikely.
The Scottish government's opponents insist the legal implications of Barroso's position will have a substantial impact on the country's status within the EU, requiring it to join the euro and EU treaties on open borders, and damage the Scottish National party's chances of winning the referendum on independence.
Barroso's interpretation was bluntly rejected by Sturgeon. She insisted there was no provision within EU law for any existing part of the EU to be removed from the union's treaties, or for its people to stop becoming EU citizens.
"The crucial point is that these negotiations will take place from within the EU, because in the period immediately following a 'yes' vote in the referendum, Scotland will still be part of the UK and the EU," she said.
"No serious person can argue that it is anything other than in the interests of the EU to keep Scotland in continuous membership, given this country's huge natural resources in energy and other aspects which make us such a valuable European partner."
The Scottish government argues that the deal signed by Salmond and David Cameron in October, to stage the independence referendum in 2014, would give an independent Scotland protected status within the EU.
It believes the wording of the Edinburgh agreement meant independence would be based on a mutually agreed process to break up the UK, and not a unilateral secession.
Further, it believes EU membership would be negotiated in parallel with the independence talks with London. It would allow Scotland to immediately inherit all the treaty obligations and opt-outs belonging to the UK, including keeping the pound and the UK's opt-outs on the Schengen agreement over border control.
The UK government argues that Salmond's interpretation misunderstands the legal purpose of the Edinburgh agreement: it simply establishes the legal framework of the referendum within UK law and makes no comment about Scotland's membership of international bodies.
On a tour of the US at the weekend, Michael Moore, the Scottish secretary, told an audience in Washington that a "downsized" independent Scotland would lose influence and status in Europe. So too would a smaller UK, but it would remain comparatively wealthier and more powerful.
"How would Scotland negotiate in good faith [to become an EU member] yet stay out of the euro when the commitment to join is a legal requirement for all new member states, given explicitly by every new member since 2005, and the plainly stated intent of Iceland and Croatia as part of their ongoing application process?"
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