Syria-born Imad al-Din Barakat Yarkas, also known as Abu Dahdah, faces a sentence of 62,512 years in jail on allegations of "terrorist murder" for helping the hijackers plan the 2001 attacks on New York and Washington.
Two others - Morocco-born Driss Chibli and Syria-born Ghasub al-Abrash Ghayun - also are accused of aiding 11 September hijacker Muhammad Atta during his travels in Spain in July 2001, when, investigators believe, he carried out the final planning for the attacks.
The trial, which started on Friday, could take months and comes after failed terrorism prosecutions in Germany and the Netherlands.
It is the first held at a high-security building remodelled for trials with multiple defendants where the suspects will sit in a bullet-proof glass cubicle.
Among those charged along with the defendants was al-Qaida leader Usama bin Ladin, but Spain cannot try suspects in their absence.
The suspects include Aljazeera
journalist Taysir Alluni
The case was prepared by Spanish Judge Baltasar Garzon, who has been investigating Muslim militancy since 1991 - long before train bombings by radicals killed 191 people in Madrid on 11 March 2004.
Garzon has since forwarded the case to trial judges, and the High Court's top criminal judge, Javier Gomez Bermudez, will preside over a three-judge panel hearing the case.
All the suspects but one are charged with belonging to a terrorist group, and that includes Taysir Alluni, a reporter for Aljazeera, who interviewed Bin Ladin shortly after the attacks on New York and the Pentagon.
"I am innocent. Whatever the outcome of the trial, I will maintain that until death," Alluni said.
The Spanish court ordered the temporary release and lifting of house arrest on Alluni, Aljazeera's correspondent in Madrid Michael Al-Kik reported.
He has been under house arrest for medical reasons while the others have been held in jail.
Alluni will testify before the court on 11 May to defend himself against charges of belonging to al-Qaida.
"The judge's case always refers to circumstantial evidence, evidence taken from telephone calls in Arabic which were badly translated and even more badly interpreted by the police," Alluni said.
The only person convicted so far in relation to the 11 September attacks is Morocco-born Munir al-Mutassadiq, who last year won an appeal against his conviction in Germany and is being tried a second time.
In the United States, accused 11 September conspirator Zacarias Moussaoui plans to plead guilty and admit a role in the attacks, US government sources said on Wednesday.