Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g,, ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C, boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47 Delta) was analyzed. Depletion of Pmp47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol, Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47 Delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases, Methanol-induced cells of pmp47 Delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47 Delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes, Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus, We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47 Delta and M13-pmp47 Delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus, We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes, Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47 Delta and M13-pmp47 Delta strains, Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.