Peroxisomes are massively induced when methylotrophic yeasts are cultured on methanol as the sole carbon and energy source. An analysis of the protein composition of the peroxisomal membrane and the generation of probes against two peroxisomal membrane proteins (PMPs) have been undertaken. Peroxisomes from Candida boidinii were obtained from sucrose gradients as previously described or from a novel one-step purification of the organelle on a Percoll gradient. The protein composition of the membranes from these two preparations was virtually identical. About 10 proteins comprise nearly all of its protein mass. The most prominent proteins have molecular masses of 120, 100, 47, 31-32 (a triplet), and 20 kDa; significant amounts of alcohol oxidase and dihydroxyacetone synthase, the two abundant matrix proteins, also remain associated with the membrane. Glycosylation of the membrane proteins could not be detected. Exposure of the membrane to chaotropes shows that PMPs 100 and 20 are the most easily removable, whereas PMP 47 appears to be the most tightly associated. Mice were injected with peroxisomal membrane, and hybridoma lines were isolated that produced antibody against PMP 20, PMP 47, and dihydroxyacetone synthase. Indirect immunofluorescence with these monoclonal antibodies confirmed that all three proteins are localized to the peroxisomal cluster. Immunoblotting experiments demonstrated that peroxisomal membrane as well as matrix proteins are induced by methanol.