The Acyl-CoA oxidase (AOX) isozymes catalyze the first steps of peroxisomal beta-oxidation, which is important for the degradation of fatty acids. Using conserved blocks in previously identified yeast POX genes encoding AOXs, the authors have shown that five POX genes are present in the yeast Yarrowia lipolytica. These genes show approx 63% identity among themselves, and 42% identity with the POX genes from other yeasts. Mono-disrupted Y. lipolytica strains were constructed using a variation of the sticky-end polymerase chain reaction method. AOX activity in the mono-disrupted strains revealed that a long-chain oxidase is encoded by the POX2 gene and a short-chain oxidase by the POX3 gene.