The driver of a Nissan Note, a 79-year-old woman from Bury, suffered serious internal injuries and was taken to Royal Preston Hospital after the crash at around 12.05pm.
Her passenger, an 83-year-old woman from Radcliffe, suffered a wrist fracture, whilst another driver - a 25-year-old woman from Barnoldswick, suffered whiplash injuries.
Police say the 79-year-old Nissan driver is believed to have joined the motorway by driving the wrong way onto the exit slip-road at Junction 3.
After reaching the top of the slip road, the pensioner reportedly tried to turn hard left in front of oncoming traffic travelling westbound towards Preston.
Her Nissan then smashed into an oncoming Fiat 500, leading to an emergency response that shut the stretch of motorway between junctions 3 (Blackburn West, A674) and 4 (Darwen / Blackburn South, A666) for five hours.
Appeal for witnesses and dash-cam footage
Sgt Finn Quainton, of Lancashire Police’s Tactical Operations, said: "We are appealing for information following a serious injury collision close to Junction 3 of the M65.
"The motorway is likely to have been very busy and I believe many people, travelling both westbound and eastbound, will have seen what happened.
"If you have any information, or dashcam footage showing what took place, please get in touch."
Anyone with information can contact police on 101 or email [email protected] quoting log 0544 of September 2.
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