In an interview with ABC World News to be aired Monday night, Ahmadinejad said "yes" when asked if he would do his best to set them free.
But, he added, "How do you know they have accidentally crossed into Iran? How do you know they were looking for waterfalls and forests?"
It was a bit of a contrast to an interview with The Associated Press in September, when Ahmadinejad said he would ask the judiciary to "look at the case with maximum leniency."
Josh Fattal, 27, along with Shane Bauer, 27, and Sarah Shourd, 31 — all graduates of the University of California at Berkeley — had been trekking in Iraq's northern Kurdistan region when they accidentally crossed the border, according to their families.
Last month, Ahmadinejad said the United States was holding several Iranian citizens, raising concern that his government might be seeking to use the Americans in a deal.
Meanwhile, Iran's foreign minister said Monday he expects a speedy trial for three American hikers.
Foreign Minister Manouchehr Mottaki declined to specify what charges the Americans faced, but Iran's chief prosecutor said last month the Americans are accused of spying.
"These Americans entered Iran illegally and with aims that arouse suspicions and doubts," Mottaki said at a press conference in Beirut on Monday.
"We expect appropriate sentences to be issued very soon," he added.
The hikers' parents have appealed for their children's release.
Wearing a headscarf, the mother of one of the hikers appeared last week in a video asking the Islamic Republic's supreme leader to release her daughter and her friends for the holidays.
"Sarah, Shane and Josh are good people," Nora Shourd of Oakland, Calif., said in the video posted on a Web site dedicated to freeing the three. "They meant no harm to the Islamic Republic of Iran and respect your ancient and noble civilization. If they entered Iran, it was an innocent mistake."
The Americans' jailing comes amid a bitter standoff between the U.S. and Iran over Tehran's disputed nuclear program.
The U.S. has no diplomatic relationship with Iran. Since the three were captured, the only word on their condition came after two prison visits by Swiss diplomats, who represent U.S. interests in Iran. The last prison visit was at the end of October, with the diplomats reporting the three appeared healthy.
The Associated Press contributed to this report.