"Could you tell me please what has transpired?" Winfrey asked Letterman during the show. "I have never for a moment had a feud with you."
It was Winfrey's first guest appearance on "The Late Show" with Letterman, although she twice appeared on his NBC show before the comic moved to CBS in 1993.
While presenting Letterman with a gift, she alluded to his much-maligned joke as an Academy Awards host in 1995 — the awkward "Oprah, Uma. Uma, Oprah" introduction.
The gift, wrapped in purple to coincide with the opening of the Winfrey-produced Broadway musical "The Color Purple," was a signed, framed photograph of herself and Uma Thurman.
"I want you to know, it's really over, whatever you thought was happening," Winfrey said.
Letterman, seeing the photo, responded: "Are you sure it's over?"
Letterman has frequently joked about Winfrey through the years, and he devoted plenty of time this week to hyping the Thursday appearance. In 2003, Winfrey told Time magazine she wouldn't go on his show because she's been "completely uncomfortable" as the target of his jokes.
"I can't thank you enough," Letterman said Thursday. "It means a great deal to me and I'm just very happy you're here."
"Does it really?" asked Winfrey. "I've been hearing for the past week you talking about it, and I didn't know if you were really serious or you were just doing your `Dave thing."'
But Letterman proved his intentions were earnest, discussing in-depth Winfrey's efforts to lend support to communities in Africa and the good intentions of her syndicated program, "The Oprah Winfrey Show."
"You have meant something to the lives of people," Letterman said. "We're just a TV show."
The approach clearly caught Winfrey off guard, as she repeatedly exclaimed, "I can't believe you're being this serious!"
"What do you want, Tony Danza?" quipped Letterman, alluding to another fellow talk show host who waited in the wings as a "stand-in host."
To cap off his gallant reception of Winfrey, Letterman escorted her during the show taping to the premiere of "The Color Purple," which opened Thursday across the street at the Broadway Theater.
Before walking her out, Letterman said, "I think we'll just pencil you in for the next 16 years."