Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5′-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5′-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5′-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5′-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5′-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5′-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3′ of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5′-flaps from either pre- or post-repair substrates.