The 29-year-old also equalled the seven-under-par 54-hole scoring record at Oakmont set by eventual winner Ernie Els in 1994, with Americans Dustin Johnson and Andrew Landry his nearest challengers on three under.
Lee Westwood and Daniel Summerhays were a shot further back, with South Africa's Branden Grace the only other player under par after completing a 66 on Saturday evening.
Lowry, who called a penalty on himself during the second round when his ball moved as he addressed a birdie putt on the 16th, holed from nine feet for birdie on the 15th and five feet for another on the 17th after a superb pitch from heavy rough.
The world number 41 - coincidentally the same ranking as Angel Cabrera when the Argentinian won at Oakmont in 2007 - was in danger of dropping a shot on the 18th when he left a long birdie attempt 10 feet short, but calmly holed for par before punching the air in delight.
"To be honest I would have taken four pars and gone home for a little rest, " said Lowry, who won the WGC-Bridgestone Invitational in Akron last year, just 115 miles from Oakmont.
"I hit a big drive down 15 and only had a nine iron in there, hit it pretty close and made that, hit a lovely pitch on 17 and a really good save on the last.
"It was a really good morning's work and that was one of the best rounds of my career, a 65 on this course. I just need to try stay in the moment, stay patient and see what happens.
"I've not have many good results this season but feel like I have been playing some of the best golf of my career and hopefully I can keep going and you never know what can happen."
A victory for Lowry on Sunday would make it 10 major titles for Irish golfers in the last 10 years after the successes of Padraig Harrington, Rory McIlroy, Darren Clarke and Graeme McDowell, although the 29-year-old from Offaly would be the first from the Republic of Ireland to win the US Open.
Asked if he believed he could add a major to the WGC-Bridgestone Invitational title he won last year, Lowry told Sky Sports 4: "I feel like my game's good enough and there's no reason I can't do that if I play well this afternoon.
"I've never felt so comfortable on a course in my life. I'm not sure it's going to be the same story on the first tee this afternoon but I am where I am and I've worked hard to get here."
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