A cell-free extract of methanol-grown Pichia methanolica cells was found to contain nine alcohol oxidase (AOD) Isozymes by active staining of a native polyacrylamide electrophoresis gel. Our previous study revealed that AOD in P. methanolica was encoded by two genes, MODI and MOD2, and the results of an experiment involving Candida boidinii as an expression host suggested that the AOD isozymes observed in P. methanolica were due to random association of Mod1p and Mod2p into an active octamer [Nakagawa et al., Yeast, 15, 1223-1230 (1999)]. This study was conducted using P, methanolica MOD1- and/or MOD2-gene disrupted strains to confirm a previous hypothesis. While the cell-free extract of the wild-type strain gave nine ladder bands, the mod1 Delta and mod2 Delta strains gave a single active AOD band corresponding to the mobilities of Mod2p and Mod1p on a native electrophoresis gel, respectively. The cell-free extract of glycerol-grown wild-type cells gave a single band corresponding to Mod1p, showing that only MOD1 is expressed in glycerol-grown cells. While the expression of both MOD1 and MOD2 was induced by methanol, this finding and our previous observations indicated that the expression of MOD1 and MOD2 was controlled by a distinct regulatory mechanism in P. methanolica.