Three men believed to be in their late teens and early 20s died after their car and an articulated lorry collided.
A third person was taken to hospital with serious injuries following the crash on the A10 near Turnford yesterday, a spokesman for Hertfordshire Police said.
The accident happened on the northbound carriageway at around 2.15pm when a Vauxhall Corsa collided with the Mercedes lorry.
The driver and two passengers in the Corsa died at the scene while a third passenger was taken to the Royal London Hospital, Whitechapel, by air ambulance with serious injuries. The driver of the lorry was not injured.
Ambulance crews from Cheshunt, Melbourn and Welwyn Garden City attended the scene while the Hertfordshire Air Ambulance was also sent.
East of England Ambulance Service NHS Trust spokesman Gary Sanderson, who attended the scene, said: "First and foremost our thoughts are firmly with all of the families of the men involved in this tragic incident.
"Following a rapid scene assessment by paramedics at the scene, three men were pronounced dead shortly after. A fourth man, who was seriously injured, was treated by land and air ambulance crews, before he was flown to the Royal London Hospital in Whitechapel for further care."
Sgt Colin Jenkinson of the roads policing unit said: "Three men have tragically died and one man has been seriously injured following this collision and we are providing support to their families at this difficult time.
"We are conducting a thorough investigation into the circumstances of the collision and are appealing for anyone who may have witnessed the incident to get in contact as soon as possible.
"Perhaps you saw the vehicle just before the collision took place or maybe you saw something immediately after. Any information you provide could be crucial to our investigation."
Anyone with information is asked to contact police on 101.