The clip sees Letterman place a personal call to Winfrey at Lohan's request, and pretend to pose as the actress's secretary, reported Ace Showbiz.
Winfrey is heard questioning Letterman about who he actually is, before the comedian reveals the prank.
Lohan also broke down in tears when Winfrey spoke to Letterman about the progress that the 'Mean Girls' star has made since leaving rehab.
"I think she's doing okay," Winfrey said of Lohan. "We're really pleased that she's making some progress.
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