In this study, we describe the molecular characterization of the PmPEX14 gene encoding the peroxisomal membrane protein from the methylotrophic yeast Pichia methanolica. The pex14 Delta strain of P. methanolica lost its ability to grow on methanol and oleate but grew normally on glucose. Disruption of the PmPEX14 caused a decrease in the activities of peroxisomal methanol-metabolizing enzymes and mislocalization of those proteins into the cytosol and vacuole. Taken together, these findings show that PmPex14p has an essential physiological role in methanol metabolism in P. methanolica. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.