Kupcho shot a 2-under 70 at rainy Golden Ocala, setting up the eagle with a fairway wood from 215 yards that just cleared the rocks and water fronting the green on the par-5 18th.
"I actually was like standing on the tee and I was like, 'I feel an eagle coming on.' So, it was pretty cool to do it," Kupcho said. "But when I got down there to the fairway I definitely didn't think it would be possible to get it close and I was not aiming anywhere close to where my ball went, but it happened to go right at the pin and carried by a couple yards, carried the water by a couple yards, and ended up in a good spot. I was able to just make the putt."
Ernst parred the last six holes in a 69.
"I didn't quite have it ball-striking-wise today," Ernst said. "I hit a few good shots, but then kind of sprayed a few drives and kind of had to work a little bit harder that I have had to work the last couple days."
The two-time LPGA Tour winner chipped in for birdie on the par-4 ninth. She was at 13-under 203.
"I know I need to go play well." Ernst said. "I can't not play well and win, so it's going to be the same type stuff. Obviously, sleeping on the lead the last two days I know what to expect. I've slept really well."
Kupcho is winless on the tour. The former Wake Forest star from Colorado won the 2018 NCAA title and the inaugural Augusta National Women's Amateur in 2019.
She overcame a slow start Saturday to remain close.
"It was just a tough start," Kupcho said. "The greens were really slow when it was raining and I ended up three-putting the second hole."
Laura Davies, the 57-year-old Hall of Famer who rebounded from an opening 75 with a 69 on Friday to advance to the weekend at even par, was 4 over after a 76. She's playing on a World Golf Hall of Fame exemption.
Ernst and Kupcho are in position to give the United States three straight victories to open a season for the first time since 2007.