The opposition and US said President Bashar al-Assad had no legitimacy and must step down from power.
Syria’s foreign minister had a terse exchange with the UN’s Ban Ki-moon over the length of his speech and said only Syrians could decide Bashar al-Assad’s fate.
The conflict has left more than 100,000 dead and millions displaced.
The summit is discussing the Geneva communiqué which lays out a political transition plan for Syria.
Wednesday’s initial meeting, involving speeches from 40 or so foreign ministers – has now ended. The direct talks are scheduled to begin in Geneva on Friday.
At a fractious evening news conference, during which there were repeated calls for calm, Ban Ki-moon spoke of the suffering in Syria, saying: “Enough is enough. The time has come to negotiate.”
He said that “the really hard work begins on Friday”, adding: “We have a difficult road ahead, but it can be done and it must be done.”
Ban Ki-moon dwelt on the Geneva communiqué, which calls for a transitional government in Syria, saying he was disappointed with the attitudes of both the Syrian government and its ally, Iran.
UN mediator Lakhdar Brahimi said he would speak to the Syrian government and opposition delegations separately on Thursday and that he hoped both teams would meet in the same room on Friday.
This would be the first face-to-face meeting between the Syrian government and the main opposition – the National Coalition – since the conflict began in 2011.
At his press conference, Secretary of State John Kerry stressed that the Geneva communiqué and its call for political transition was the paramount focus of the summit.
“Every delegation, with one exception, embraced the Geneva communiqué,” John Kerry said, referring to the Syrian government.
“No-one has done more to make Syria a magnet for terrorists than Bashar al-Assad,” he said.
“You cannot save Syria with Bashar al-Assad in power.”
A member of the Syrian team, UN ambassador Bashar Jaafari, criticized the exclusion of Iran from the meeting, and condemned many of Wednesday’s speeches as “provocative and repetitive statements based on hatred towards the Syrian government”.
He also accused Gulf states of “inciting terrorism” in Syria.
Syria’s Foreign Minister Walid Muallem said some states attending the talks had “Syrian blood on their hands” and called the opposition “traitors”.
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