United Nations Syria envoy Staffan de Mistura met representatives of both delegations for "informal meetings" ahead of the negotiations, due to begin on Monday.
The long-awaited talks will open on the eve of the fifth anniversary of the outbreak of Syria's civil war, in the latest bid to end the bloodshed that has killed more than 270,000 people and displaced millions.
But disagreements over the agenda have already cast a shadow over the negotiations, and on Sunday Western powers hit out at the regime for saying that removing President Bashar al-Assad would be a "red line" in the talks.
Damascus and the High Negotiations Committee (HNC), the main opposition umbrella group, have already clashed over what the talks will cover and hours before they were due to start the agenda was still up in the air.
US Secretary of State John Kerry said the remarks from his Syrian counterpart were "clearly trying to disrupt the process... (and) clearly trying to send a message of deterrence to others".
Speaking in Paris after meeting with his European allies, he also warned Damascus and its allies Russia and Iran against "testing boundaries" or destroying a fragile ceasefire that began on February 27.
"This is a moment of truth, a moment where all of us have to be responsible," he said.
French Foreign Minister Jean-Marc Ayrault went further, calling Syrian Foreign Minister Walid Muallem's comments a "provocation" and a "bad sign" in the peace efforts for Syria.
"There will be no political process if the opposition is not closely involved and confident," he added, calling on all the players in Syria's war to ensure "the peace process is sincere and real steps are taken".
While analysts say much has changed since the last round of indirect talks collapsed in February, Assad's fate and whether elections will be held within 18 months remain huge obstacles.
France, US, Russia
Experts have already cast doubt on whether the talks will get off the ground and, if they do, whether any agreement will be able to take hold on the fractured battlefields where multiple groups are competing for dominance.
Bassel Salloukh, political science professor at the Lebanese American University of Beirut, said the current talks were more of a forum for international powers involved in the conflict.
"The strategic interests of Russia and the US will determine the shape of the settlement in Syria rather than the aspirations of its peoples," he said.
Iran and Russia have been supporting Syria's regime with weapons and airstrikes, while the US, Europe, as well as Tehran's regional rival Saudi Arabia, have been backing the opposition.
Their leverage over warring parties in Syria has its limits, however, as half of Syrian territory is controlled by jihadists from the Islamic State (IS) group or Al-Qaeda affiliate Al-Nusra Front.
These groups have not been covered by the temporary ceasefire, which has also been strained by accusations of violations by the regime and rebels.