Dufner shot a 6-under 66 at the Bay Hill Club and Lodge, as did Charlie Wi, and they owned a two-shot advantage over Nick Watney.
Dufner began last week's Transitions Championship with a pair of 66s, but then faded on the weekend to a tie for 10th. He is coming off his best season as a professional, one in which he finished second in the PGA Championship and moved into the top 40 in the world rankings.
Tiger Woods, seeking his first tour victory since 2009 and hoping to boost a career plagued by personal issues and injuries, was among those in a tie for fourth after firing a 69.
Woods showed no lingering problems from a strained Achilles tendon he suffered during the final round of the WGC-Cadillac Championship two weeks ago. He withdrew from that tournament as a precaution.
Phil Mickelson struggled to a 1-over 73 and had to birdie three of his final six holes to do that.