The V/Line and Metro trains were separated on Saturday afternoon. The Metro train had been travelling east towards the city but was stopped, unexpectedly, at the Maidstone Street level crossing near Ajax Road in Altona on Friday. About 7pm, a second train, travelling from Geelong, rear-ended the suburban train at low speed. It is understood that debris on the track may have caused the Metro train to stop, according to a source.

Police were believed to have been following up reports that there was an attempt to stop the train to launch a graffiti attack.

"The driver of the Metro train had reported hearing a bang underneath the train and lost air pressure to the brakes, which applied the brakes," a source said.

"If he heard a bang the train may have run over something that had been either placed on the tracks or fallen on to the tracks."

About 60 people were assessed by Ambulance Victoria paramedics and four were taken to hospital.

There were no passengers on the Geelong train, but the driver and conductor had minor injuries.

Seven people were treated at the scene. Public Transport Victoria chief executive Mark Wild said the trains were separated more than 19 hours after they collided.

"The two trains were separated at around 2.15 this afternoon and Metro and V/Line engineers are now inspecting the vehicles to determine the best way to clear the scene," he said.

The two drivers involved in the crash were experienced and had tested negative for drugs and alcohol, a well-placed source said.

The Office of the  National Rail Safety Regulator (ONRSR) and the Transport Safety chief investigator's office were investigating at the scene.

ONRSR spokeswoman Gail Sambidge-Mitchell said that once the trains were cleared the track would need to be inspected.

"In terms of when services will resume, that is up to the operator, which is Metro, but obviously they have to inspect the track first," she said.