We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.