PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene. The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria. The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a predicted molecular mass of 112,258 Da. The predicted Pay4p sequence contains two putative ATP-binding domains and shows structural relationships to other potential ATP-binding proteins involved in biological processes as diverse as peroxisome biogenesis, vesicle-mediated protein transport, cell cycle control, and transcriptional regulation. These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP binding. Pay4p shows sequence conservation with Pas1p and Pas5p, putative ATPases required for peroxisomal assembly in the yeasts Saccharomyces cerevisiae and Pichia pastoris, respectively. Pay4p, Pas1p, and Pas5p are presumably related members of a family of putative ATPases involved in peroxisome biogenesis. Pay4p is synthesized in low amounts in Y. lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.