The attack, killing 191 people and injuring 1,824 others, constituted the worst attack in Spanish history and the deadliest in Europe since the bombing of a PamAm jet over Scotland in 1988.
Prosecutors allege that the 29 mainly Moroccan defendants were working on behalf of the Moroccan Islamic Combat Group, a North African al Qaeda offshoot.
They are seeking jail sentences totalling around 270,000 years for the seven main suspects - 40,000 years each. However, under current Spanish law the maximum sentence for a criminal act of terrorism is 40 years in prison.
The trial will take place at the same high-security court venue on the outskirts of Madrid where two years ago Spain staged the biggest trial to date in Europe involving Osama bin Laden's al Qaeda network, when the Syrian head of a Spanish-based al Qaeda cell jailed for his role in the September 11, 2001 attacks in the United States.
The trial, the first in Spain to use entirely digital technology that permits real-time, on-screen access to data in the courtroom, will run to the end of July.
Hearings will be held three days per week to accommodate some 600 witnesses and a further 107 technical experts. Verdicts are due by October. Investigators have sifted through more than 93,000 files while the charge sheet and appendages run to more than 1,400 pages.
Security sources say the security around the 180-seat courtroom, which will be packed with witnesses, officials, survivors and relatives of the victims, will be "spectacular".
An overflow room in the building will house 120 additional seats for, among others, more relatives of the victims and the survivors. A team of psychologists will be on hand to help them cope with their emotions as they watch the 29 suspects on a flat screen.
Spanish television will provide a permanent feed of the trial throughout and has offered the signal to world media. Victim compensation, most already paid out, will total 95.5 million euros (124.2 million dollars).