The Rome, which was held on the spot where the EU's forerunner was born, was also clouded by a row over the EU's incoming executive commission, which was withdrawn at the last minute, leaving the union in the hands of a caretaker team in Brussels.
The assembled leaders were urged to press ahead speedily with ratifying the constitution, which must pass referenda in at least 10 EU countries over the next two years to enter into force.
"Those decisions we cannot take for granted," said outgoing European Commission chief Romano Prodi on Friday.
"The constitution that we are signing today will therefore need to be backed up by a continually renewed commitment on the part of both citizens and governments," added Italy's Silvio Berlusconi, hosting the event.
The ceremony took place in the ornate Orazi and Curiazi hall,
where the Rome Treaty which established the European Economic Community - the EU's predecessor - was launched by six countries on 25 March 1957.
The constitution, agreed in June after two years of haggling,
aims to streamline EU institutions and prevent decision-making gridlock in a bloc which grew from 15 to 25 members this year, with several more waiting in line.
It notably foresees a longer-term EU presidency to replace the current six-month musical chairs system, while streamlining the executive commission and creating a new post of EU foreign minister.
Ciampi hailed the constitution
Italian President Carlo Azeglio Ciampi hailed the new constitution as "a turning point in the history of humanity".
"The act which you signed today distances us definitely from that chasm of tragic internecine wars; the spectres of the 1930s will no longer haunt the souls of future generations," he said, referring to the rise of Nazi Germany.
No vote fears
But the historic document has still to be ratified by all 25 EU member states to come into force. At least one or two no votes are feared in the referenda to be held over the next 48 months.
Analysts say the scale of the political problem will depend on the number of no votes and, inevitably, where they occur. A thumbs-down by Britain, for example, would fuel talk about sidelining some countries; a negative result in France could lead to a full-blown EU crisis or even implosion.
British Foreign Secretary Jack Straw said on Friday that Britain was likely to hold its referendum on the EU constitution early in 2006. Blair made no public comment in Rome, leaving shortly after the end of the ceremony.
But of more immediate concern to the EU leaders is the commission standoff.
The Rome gathering comes just two days after the European Parliament forced incoming commission president Jose Manuel Barroso to withdraw his proposed team after protests over Italy's EU nominee for the sensitive EU justice portfolio, Rocco Buttiglione.
Berlusconi's government immediately said it would not drop him, but both the Italian leader and Buttiglione are expected to come under growing pressure to "do the honourable thing".
Buttiglione, who was among dignitaries at the Rome ceremony, remained tight-lipped.
Buttiglione (L) is at the centre of
the commission spat
But Spanish Prime Minister Jose Luis Rodriguez Zapatero implicitly called for him to go. "It is limited to one person, so that's where we should start the discussion. There have to be changes," he said.
Italy has so far resisted pressure to withdraw Buttiglione, a conservative with outspoken views on gays and women, as its Brussels nominee. Foreign Minister Franco Frattini says Rome hopes for a speedy solution to the problem.
German Chancellor Gerhard Schroeder admitted that EU leaders talked about the commission row in the sidelines of the Rome ceremony, but expressed hope the standoff could be resolved in the next two weeks.
"I would not talk about a crisis - not yet. If there is an agreement in the next 14 days, which I suppose, then it will be resolved," he said.