We established a reverse-genetic approach to identify peroxisomal proteins involved in peroxisomal fatty acid metabolism of Saccharomyces cerevisiae. Putative peroxisomal peripheral membrane proteins were isolated by successive extraction of purified peroxisomes and purified by HPLC and SDS/PAGE. Six proteins were identified by peptide sequence analysis, including acyl-CoA oxidase and a trifunctional enzyme of the peroxisomal beta-oxidation system as well as peroxisomal malate dehydrogenase 3 and carnitine acetyltransferase. In addition two previously unknown putative peroxisomal proteins were identified, an unknown 40-kDa protein and a protein which we named Pcs60p, both major constituent of the isolated protein fraction. Pcs60p is encoded by ORF Z36091 of chromosome II from S. cerevisiae and consists of 543 amino acids with a molecular mass of 60.5 kDa. Biochemical, immunofluorescence microscopy and immunocytochemical data confirmed that Pcs60p is a peroxisomal peripheral membrane protein but the protein is also localized in the peroxisomal matrix. Consistent with the intraperoxisomal localization, the consensus sequence for a peroxisomal-targeting signal 1 (PTS1) is present at the extreme C-terminus of Pcs60p. Deletion studies revealed that the peroxisomal localization of the protein depends on the presence of this signal sequence. Expression of Pcs60p is highly inducible by oleic acid, however, the protein is dispensable for growth on oleic acid as single carbon source. Pcs60p belongs to the family of proteins which act via an ATP-dependent covalent binding of AMP to their substrates and shows the highest degree of similarity to the Escherichia coli long chain acyl-CoA synthetase.