Methanol oxidases (MODs) were investigated in some methanol-utilizing yeasts. Pichia methanolica IAM 12901 and IAM 12481 (= Pichia cellobiosa) produced 9 forms of methanol oxidase on electrophoresis when grown on methanol but 1 form when grown on glycerol or pectin. Other strains-Candida boidinii IAM 12875, Pichia angusta IAM 12897, and Pichia pastoris IAM 12903-produced one band on electrophoresis when grown on methanol. Methanol oxidases with the smallest and largest Pm (relative mobility)-MOD A and MOD B-were purified from methanol-grown cells; another methanol oxidase (MOD P) was purified from pectin-grown cells of P. methanolica IAM 12901. All the purified enzymes were octamers composed of subunits with a molecular mass of 70,000 Da. MOD A and MOD P were mostly identical with each other in terms of their enzymatic characteristics, but MOD B differed from the other two with respect to substrate specificity, the prosthetic group, and the apparent K-m for methanol. Southern analysis of the genomic DNA of P. methanolica LAM 12901 was carried out with the partial methanol oxidase gene as a probe. The genomic DNA of this yeast showed the presence of 2 methanol oxidase genes. It was concluded that the 9 forms of MOD in P. methanolica were isozymes of a chimera octamer consisting of 2 subunits encoded by different methanol oxidase genes.