Gary Woodland held a one-shot advantage over Rickie Fowler courtesy of a six-under 64 at Bellerive Country Club on Thursday.
The world number 44 moved to seven under with a birdie at the 11th after beginning his second round on the back nine, but a bogey at 14 dropped him back to where he started the day in St Louis.
Kisner, who finished in a tie for second at The Open having held the lead at one stage at Carnoustie, made four birdies in his opening six holes with the sun shining, moving him to the top of the leaderboard at seven under.
Brandon Stone held a share of top spot with Woodland and Kisner after hitting the turn in 33, but a dropped shot at the first - his 10th hole of the day - saw him slip back.
Open champion Molinari, meanwhile, made three gains in his opening five holes to raise hopes of winning back-to-back majors after claiming the first of his career at The Open.
World number one Dustin Johnson was level par for his round at the turn after failing to hold a 10-foot par putt at 15, having missed the fairway, before moving back to three under with a first birdie of the day at 18.
Adam Scott went out in 31 to move to four under, while Brooks Koepka also made a promising start to sit alongside Johnson, Justin Rose and Marc Leishman a further shot back in the chasing pack.
Fowler, eyeing an elusive maiden major, goes out at 1:04pm local time (18:04 GMT).
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