As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol‐utilization‐defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol‐ and ethanol utilizing‐deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome‐deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.