The Pas1 protein (Pas1p) is required for peroxisome biogenesis in Saccharomyces cerevisiae and contains two putative ATP-binding sites, each within a domain which is conserved among members of the recently characterized AAA-family. To elucidate whether both putative ATP-binding sites are essential for Pas1p function, lysine 467 of the first and lysine 744 of the second putative ATP-binding site were each changed to glutamate by site-directed mutagenesis. While replacement of lysine 744 abolished the function of the Pas1 protein in peroxisome biogenesis, replacement of lysine 467 had no obvious effect.