To examine whether the hepatic aldehyde oxidases of male and female mice, which exhibit sex-related differences in benzaldehyde oxidation, are qualitatively different, we purified the enzymes from untreated male and female ddy mice and testosterone-pretreated female mice by sequential chromatography of benzamidine-Sepharose 6B and DEAE-5PW columns and characterized the enzymes. Purified enzymes from untreated male and female mice and from testosterone-treated females gave a single protein band at M(r) 265K and M(r) 138K on native and SDS-polyacrylamide gradient gel electrophoresis, respectively. The susceptibilities of the enzymes from both sexes to inhibitors such as menadione, norharman, quinacrine, estradiol, and SKF 525-A were also indistinguishable. However, the K(m) values for benzaldehyde, p-dimethylaminocinnamaldehyde, and 2-hydroxypyrimidine were two- to fourfold higher in the untreated female enzyme than in the untreated male enzyme, while the testosterone-induced female enzyme showed K(m) values similar to those of the male enzyme. These results indicate that the hepatic aldehyde oxidases of the sexes are quite similar with respect to molecular size and susceptibility to inhibitors, but their kinetic properties are somewhat different, suggesting the existence of microheterogeneity in sex differences. It also suggests that treatment of female mice with testosterone might induce the male-type enzyme.