Saccharomyces cerevisiae mutants lacking the structure-specific nuclease Rad27 display an enhancement in recombination that increases as sequence length decreases, suggesting that Rad27 preferentially restricts recombination between short sequences. Since wild-type alleles of both RAD27 and its human homologue FEN1 complement the elevated short-sequence recombination (SSR) phenotype of a rad27-null mutant, this function may be conserved from yeast to humans. Furthermore, mutant Rad27 and FEN-1 enzymes with partial flap endonuclease activity but without nick-specific exonuclease activity partially complement the SSR phenotype of the rad27-null mutant. This suggests that the endonuclease activity of Rad27 (FEN-1) plays a role in limiting recombination between short sequences in eukaryotic cells.