In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut(-)) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of pel mutants, termed Pim(-), are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of pel mutants that were observed to be devoid of any peroxisome-like structure (Per(-)). In each Per(-) mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim(-) and Per(-) mutant collections involved mutations in 14 different PER genes. Two of the genes, PERS and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant pel strains and wild-type H. polymorpha were Mut(-) and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.