The three cyclists were riding from Woodend to Macedon, when a white Holden travelling in the same direction struck them from behind.

Six years ago, a bike lane was installed on the stretch of road where the cyclists were hit but it was removed amid community opposition.

Despite the lane's removal, Macedon Ranges Cycling Club president Brett Ellis, who has cycled on local roads for a decade, said it was not a dangerous spot.

"It's one of those roads where people should be able to slow down momentarily, move over and go past cyclists without any problems," Mr Ellis said.

"On a road like that there's no reason for [a crash]."

Police say the riders "wouldn't have known the driver was coming".

Officers are investigating whether speed, driver distraction, alcohol or drugs played a part in the crash, on Black Forest Drive at about 10.45am on Monday.

Detective Sergeant Mark Amos, who is investigating the crash, said another car had overtaken the cyclists safely, just moments before the crash.

"One car ahead of the offending vehicle actually overtook them safely, and then the second one drove straight into them," he said.

"The cyclists wouldn't have known the driver was coming. It's a really horrible scenario."

A St Albans man, aged in his 40s, is in a critical condition at Austin Hospital after suffering severe spinal and neck injuries.

Detective Sergeant Amos said the cyclist was "in the fight of his life to try and survive" after the crash.

Another St Albans man, aged in his 20s, remains in a stable condition at Royal Melbourne Hospital. The third cyclist was treated at the scene and taken to hospital as a support to the others.

The driver of the Holden, a 26-year-old Parkville man, was interviewed on Monday and Detective Sergeant Amos said the man was released later that day, pending further investigation.

Over the years there has been some debate about the condition of Black Forest Drive, a popular route among local and visiting cyclists.

A bike lane was installed on the road in 2011, reducing it from four car lanes to two, but it was scrapped by the state government only months later, due to community opposition.

Mr Ellis said the road was in a good condition and the focus for cyclists and the state government should instead be around enforcing a one-metre rule between riders and cars.

"It was installed, and then it was de-installed because a lot of people including cyclists complained about it," Mr Ellis, who has cycled on Macedon roads for a decade, said.

"It benefited a few people but it's not the ideal situation. I think the 'metre matters' rule is really what we need to encourage because all cyclists benefit from that."

However the Bicycle Network argued that adding bike lanes was the safest option for all road users.

"For decades now, all over the world, bike lanes have been proved to reduce crashes, not only for riders, but for cars," the group said, when lobbying for the bike lanes in 2010.

Mr Ellis said, as well as educating motorists of the danger of getting too close to cyclists, there needed to be legislation to avoid these crashes, including allowing cars to cross double white lines to avoid cyclists.

Police are investigating the cause of the crash and urge witnesses to call Crime Stoppers on 1800 333 000.