The defendants stand accused of carrying out political activities to back Eta, blamed for the deaths of 829 people in a four-decade campaign for an independent Basque homeland in northern Spain and southwest France.
Just before their trial began at a court in San Fernando de Henares, about 40 kilometres (25 miles) northeast of Madrid, the 35 accused posed for photographers behind a banner that read: "No more political trials".
"It is clearly a political persecution trial since the serious crime that the 35 are accused of is of having carried out political work," one of the accused, Pernardo Barrena, the spokesman of Basque separatist party Sortu, told reporters.
Two French nationals, Aurore Martin and Haizpea Abrisqueta, are among the accused along with veteran Basque independence leaders such as Juan Jose Petrikorena.
"We are very proud of our political activism and we think the work done has a lot to do with the new context of peace which we know in the Basque Country," Martin said as she read a short declaration before the start of the trial which is expected to end in March 2016.
Most of the accused are suspected members of Batasuna, which was banned in Spain in 2003 because it refused to condemn violence by ETA.
It was the first time since the death in 1975 of dictator General Francisco Franco that a political party has been banned in Spain.
Batasuna was authorised in neighbouring France until it voluntarily dissolved in 2013.
The 35 are charged with "participation in a terrorist organization" because of their political activities which included giving press conferences and signing articles after Batasuna was banned.
If they are convicted, they face prison terms of up to ten years as well as a ban on holding public office for at least a decade.
Their defenders consider the trial to be "anachronistic" since many of the accused are members of Sortu, a legal party created in 2012 which accepts democratic rules and seeks peace in the Basque region.
Their trial was postponed several times, most recently in January, after three of their six lawyers were arrested, accused of tax evasion, money laundering and of indoctrinating ETA prisoners.
Eta - or Euskadi Ta Askatasuna, which means Basque Homeland and Freedom in Basque - in October 2011 declared a "definitive end to armed activity" but it has not formally disarmed nor disbanded as the Spanish and French governments demand.