Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes. Its human homolog, Pmp34, is also known. In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C. boidinii (strain pmp47 Delta). The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth. The pmp47 Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate, The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47 Delta were lower than the respective level in the wildtype strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47 Delta were indistinguishable from the respective level in the wild-type strain. When lauroyl-CoA was provided in the cytosol of strain pmp47 Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47 Delta on laureate was recovered to the level of growth of the wild-type strain. Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase, These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47 Delta cells to grow on laureate. In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47 Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1(GFP-AKL).