Police found the car on June 13 in the town of Alcala de Henares, the departure point of three of the four trains bombed in the March 11 attack, a police spokeswoman said on anonymity.
She said that investigators believe the bombers used the car, a Skoda Fabia, to transport some of the explosives used in the blasts, which killed 191 people and injured 2,000.
DNA tests on clothes found in the trunk confirmed that it was used by two suspected Islamic terrorists, one of whom later committed suicide to evade capture, she said.
"Apart from clothes, an alarm clock, an agenda and a Palestinian headscarf, police spotted two cassettes with calls to jihad and martyrdom," the spokeswoman said.
The car was parked just 30 yards from the spot where a white van also believed to have been used by the bombers was found, the Madrid daily el Mundo reported Saturday. Police found it after a resident complained the vehicle had been abandoned, the report said.
The van contained clues that provided the first suggestion of Muslim involvement in the attacks.
Upon inspection, police discovered the car had been stolen and notified the rental company Hertz, whose staff took it away and started cleaning it before they found a suitcase inside.
But police believe other evidence in the car (like fingerprints) may have been destroyed in the cleaning, El Mundo said.
Of 50 people arrested in the case, 16 remain in jail, including two believed to have put backpacks stuffed with dynamite and shrapnel on trains.