Investigators are focusing on the failure of a German-made automatic train protection system and possible errors by the rail control centre in Madrid.
Recordings of radio communications between controllers and train drivers suggest that a high-speed train was instructed to resume normal operations despite the safety system remaining out of service and another train standing on the tracks ahead.
The crash occurred on Wednesday morning near the town of Urda, about 130km south of Madrid, when a train operated by state-owned rail company Renfe crashed into the rear of a stationary train belonging to private operator Iryo.
The death toll rose to 69 overnight after another body was recovered, Castilla-La Mancha regional president Emiliano García-Page said.
He said he expected no further victims to be found.
Of the nearly 200 people injured, 18 were still in intensive care units at various hospitals on Thursday.
King Felipe VI and Queen Letizia visited the injured at Toledo University Hospital, where they spoke with doctors and relatives.
Felipe said the investigation must establish why the disaster occurred and measures must then be taken "to ensure that something like this does not happen again."
Spanish Transport Minister Oscar Puente described the decision to allow the Renfe train to continue its journey as "inexplicable" and "wrong."
According to Spanish media reports based on radio recordings, the control centre in Madrid instructed the Renfe driver to "resume normal operations" despite the automatic train protection system remaining out of service following a computer failure.
The driver sought confirmation, asking: "Normal operations?" The controller replied: "Yes."
The other train was stopped around 25km further along the line.
Public broadcaster RTVE reported trains are permitted to travel at speeds of up to 200km/h on the section of track.
However, El País newspaper, citing government sources, said the Renfe train should have been instructed to proceed at a maximum speed of 40km/h and only as far as the driver could see the track was clear.
The driver of the Iryo train had also been instructed to resume normal operations, according to the reports.
She informed controllers, however, the system restart had failed and she was unable to continue.
"That's why I'm still stopped before the signal at the entrance to Urda," she reportedly said.
The control centre responded: "All right, then as soon as possible. And if there are any problems, give us a call."
By that point, the Renfe train was already moving again. It crashed into the stationary Iryo train about five minutes later.
Investigators have yet to determine whether, and to what extent, human error contributed to the collision.
As investigations begin, a judge in Toledo barred anyone not involved in emergency, security, healthcare or judicial work from the crash site with a 2km exclusion zone in both directions along the Madrid-Seville high-speed rail line.
with AP and DPA
