Rose, who trails Harrington by 657 (US$948) for the European Tour money title, nailed the ace with a 6-iron at the third hole. The Englishman also had three birdies and four bogeys at the Valderrama course.
"(It) was surreal," Rose said of his sixth career hole-in-one. "At the top of its flight I said 'go' and when it landed I said 'in' and it disappeared. Yeah, it was good."
Harrington recovered from being 4 over after the front nine to hole five of his six birdies and shoot 71.
Harrington would have finished level with Rose if not for his putting routine. The Irishman was given a penalty stroke at No. 7 when his ball moved after he had grounded his putter.
"I thought that maybe one day it's going to cost me a shot. But I'm not going to change my routine," he said. "These things, they happen."
Graeme McDowell of Northern Ireland played the swirling winds best to head the field with a 3-under 68, leading 2005 champion Paul McGinley of Ireland by one stroke. The win reached speeds of 25 miles per hour (40 kilometers per hour), and is expected to pick up for Friday's second round.
"If this wind keeps up, it's going to be a real grueling weekend," McDowell said. "I'm just content with the way I controlled the ball. Certainly 3-under par is beyond sort of what I was hoping for today."
Peter O'Malley of Australia (70) was the only other player in the 55-man field to finish under par.
Order of Merit leader Ernie Els is 217,295 (US$313,892) in front of last year's winner, Harrington, but the South African is playing in Singapore this weekend on the Asian Tour.
Rose, who was hooked up to an intravenous drip before his round due to a virus, was at 3 under by the fifth before bogeys at the seventh and ninth dropped him back to 1 under.
At the 547-yard 11th, the Englishman pulled his second shot into the left rough but recovered by sticking his wedge from 86 yards to within 15 inches for a birdie.
After three-putting for bogey at No. 14, Rose sank his birdie at the par-3 15th but missed a par-saving putt at the last.
"When I saw guys at 3 under and 2 under, I thought the course was playing even harder than that," Rose said. "I would have been ecstatic with even par."
Harrington stuck his approaches at the 10th and 11th close for easy birdies, and then curled in birdie putts at No. 16 and 17 from about seven feet to climb back from being six shots behind Rose after five holes.
"I think he helped me, no question about it. The fact that he was 3 under par through eight, nine holes ... certainly showed me that it was possible to do it," the British Open champion said. "He hit some good shots and no question, that was very positive affirmation to me that it could be done."
Swedish players Niclas Fasth (75) and Henrik Stenson (76) are also in contention for the Order of Merit, while defending champion Jeev Milkha Singh of India shot a 5-over 76.