The A9 was shut in both directions after a lorry and a car collided south of Dalwhinnie in the early hours of yesterday morning.
One man was said to be in a life-threatening condition, one is in a serious condition while the other three were not seriously injured. The man driving the lorry was not hurt.
Police Scotland said casualties were taken from the scene by helicopter and ambulance.
Meanwhile, it has emerged a cyclist involved in a collision on the road last month has died in hospital.
No official diversion was put in place after yesterday's crash and motorists faced a 152-mile detour that added four hours on to journey times.
Police asked for patience while emergency services carried out an investigation into the cause of the collision.
The road reopened just before 9am, almost nine hours after the crash.
The Scottish Ambulance Service said two people were taken by helicopter to Ninewells Hospital in Dundee. One person was taken to the hospital by road ambulance and another two people were taken to Raigmore Hospital in Inverness.
The A9 has long been regarded as Scotland's most deadly road and has been the scene of a number of fatal crashes down the years.
Between 2006 and 2010, it claimed more lives than any other road in Scotland with a death toll of 67, while there were 58 fatalities between 2008 and 2012, an average of slightly more than 14 a year.
Last October, a 47-year-old van driver was killed in a crash on the road when his vehicle struck the back of a Volvo flatbed lorry which was queuing in traffic.
And, in November, Invergordon Academy pupil Hollie Mackay, 15, was struck by a car when she got off a bus in the dark near her home village of Kildary, Easter Ross.
In October the Scottish Government announced that £18.5 million is to be spent on safety works and maintenance on the A9 during the next two years.
Work to dual its entire 269-mile length, from Dunblane to Thurso, is also scheduled to begin in 2015, although it will not be completed until 2025.
Meanwhie, police yesterday named the cyclist, who died in hospital nine days after he was involved in a collision with a coach, as 53-year-old Robert Don, from Perth.
Mr Don suffered a serious head injury in the crash on December 30 and was taken to Ninewells Hospital in Dundee.He had been in the intensive care unit until his death on Wednesday afternoon.
The collision on the A9 involved a blue-and-yellow single-decker coach on a scheduled journey between Inverness and Edinburgh. It stopped immediately and the driver and 19 passengers were not injured.
Police Scotland have appealed for witnesses to the incident which happened on the southbound carriageway of the A9 between Luncarty and Inveralmond near Perth.
A spokesman said: "Officers are still trying to trace witnesses to the incident a number of whom may have stopped to assist in the immediate aftermath."
A report will be submitted to the procurator-fiscal.