Dufner shot a 7-under 63 at the Oak Hill Country Club, breaking the course record by a shot and recording the 26th such score ever posted at a major championship.
Johnny Miller shot the first of those 63's to win the U.S. Open 40 years ago and although the likes of Jack Nicklaus, Tiger Woods and Gary Player have equaled the mark through the years, no one has been able to break what is one of the most significant records in the annuls of the sport.
Dufner reached 7-under for the day by holing a six-foot birdie putt at the par-4 16th and a birdie on either of the final two holes -- the two hardest tests on the course -- would have given him the lowest 18-hole total in the history of major championship golf.
He had two excellent chances to shoot a 62, first missing a 20-foot birdie attempt at the 17th that barely slipped by on the left side of the cup. Dufner then put his second shot at the 18th hole to within 12 feet of the hole, but his uphill putt at history came up a foot short.
"There was a lot of pressure," said Dufner, who lost the PGA title to Keegan Bradley in a playoff two years ago. "I guess I felt it."
Nevertheless, his two-day total of 9-under 131 put him two strokes in front of Matt Kuchar and first-round co-leaders Adam Scott and Jim Furyk. Kuchar shot a 66 while both Scott and Furyk had a 68. Scott, who won the Masters in April, is trying to become the first player in five years to win two majors in the same season.
U.S. Open winner Justin Rose and Henrik Stenson were tied for fifth at 134 while Steve Stricker and Robert Garrigus finished at 135. Webb Simpson, last year's U.S. Open champ, was among those at 136. Simpson shot a 64 that briefly tied the course record before Dufner did even better.
Tiger Woods and Phil Mickelson, the world's Top 2 players who shared the spotlight coming into the tournament after having pulled off significant achievements in recent days, left themselves needing a sensational round on Saturday to climb into contention.
Woods, the winner of last week's Bridgestone Invitational, shot a 70 for a 141 total that tied him for 39th place. Mickelson, who captured the third leg of a career grand slam last month in Scotland, was another shot back after his second straight 71.
Rory McIlroy, ranked third in the world, birdied four of the last seven holes to tie Woods at 141.
Dufner's round was highlighted by his second shot at the par-4 second, which landed about 15 feet past the hole and spun back into the cup for an eagle. After a light rain had fallen on the early starters, Dufner was able to take advantage of the soft greens to birdie the par-5 fourth and par-4 fifth and made the turn in 4-under.
Birdies on the 11th, 13th and 16th gave Dufner his shot at the history book.
Dufner burst on the golfing scene with his near-miss at this event in 2010, then shot up the world rankings last year when, in less than two months, he won in New Orleans, got married, won again at the Byron Nelson Classic, finished second at the Colonial Invitation and wound up tied for fourth at the U.S. Open.
Celebrity Couples of 2014 [PHOTOS]