Two carriages derailed and crashed in a tunnel near Jesus station, forcing rescuers to evacuate 150 passengers.
Officials quickly ruled out terrorism as a cause of the crash. They say high speed and a collapsing train wheel are among the likely causes.
Some 47 other people were injured, with 12 remaining in hospital, in one of Spain's worst accidents of its kind.
Two of those in hospital were reported to be in a "very critical" condition.
Earlier reports said the train driver and a pregnant woman were among those seriously injured.
Painstaking work continued after sunset as forensic experts and rescuers worked at the scene of the accident.
No final casualty figure was available, because recovering and identifying bodies from amid the wreckage was proving difficult, regional government spokesman Vincent Rambla said.
There were people with heads bleeding, cuts and bruises quite seriously injured
Graham Moore
Eyewitness
In pictures: Valencia crash
Special units trained in disaster management were deployed in central Valencia.
Those injured were taken to five hospitals around Valencia.
Briton Graham Moore, who lives in Valencia, described a "chaotic and confusing" scene 15 minutes after the accident.
"There were lots of police running around, kids with their parents. There were people with heads bleeding, cuts and bruises. The injuries appeared to be just head, neck and chest injuries - they were quite seriously injured."
Spain's Prime Minister Jose Luis Rodriguez Zapatero, was expected to cut short a visit to India to attend funerals in Valencia's cathedral on Tuesday evening, officials said.
Spanish King Juan Carlos was also expected to travel to the city, which has declared three days of mourning.
Investigations begin
Local officials and emergency services suggested speeding and defective wheels were likely causes of the crash.
Spain's national police service sent five specialist accident investigation officers from Madrid to Valencia to help establish why the train derailed.
"It seems this unfortunate accident was caused by excess speed and a wheel breaking just before it entered the station," government spokesman Luis Felipe Martinez said.
The four-carriage train was travelling on Valencia's Number One underground route, close to a junction with another line, and came off the rails between Plaza de Espana and Jesus stations.
The train had passed a safety inspection just one week before the crash, Spain's EFE news agency reported.
Carriages were busy as the accident occurred shortly after 1300 (1100 GMT) when many would have been heading home for lunch.
The emergency services were alerted by a phone call from a trapped passenger, Spanish media reported.
Last September three underground trains collided in Valencia on the same line, injuring 29 people.
The accident comes days before Pope Benedict XVI is due to visit Valencia, with preparations being made for hundreds of thousands of pilgrims to use the city's public transport network.
A Vatican spokesman said the Pope was quickly informed of the Valencia accident, and had offered prayers for the victims and their families.
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At least 43 people have died and at least 46 injured, 4 of whom are in critical condition, in a metro train crash in Spanish city of Valencia, according to the regional government. 

It has been reported that two of the carriages have derailed and overturned, leading to the evacuation of 150 passengers. 

A part of the wall in the tunnel between the Plaza de España and Jesús stations in the southwestern neighborhood of Patraix may have collapsed, causing the derailment of one of the carriages, which in turn caused the carriage behind it to overturn.

Another theory put forth by subdelegate of the regional government Luis Felipe Martínez is that the wheels on one of the carriages fell off while the train was speeding. The same source has also confirmed that there are two overturned carriages.

The fire department, paramedics and local police are on the scene after a passenger in the crashed train called the emergency services at about 1:03 p.m. CEST (1103 UTC). The authorities have closed the area to all road traffic and routes 1 and 2 of the Valencia metro system, which use the affected rail tracks, are shut down.

The incident comes days before Pope Benedict XVI was due to visit Valencia for the World Meeting of the Families, expected to be attended by one million pilgrims.