The sex differences in in vitro metabolism of testosterone in the homogenates, the microsomes or the 105,000 × g supernatant fluid of adult rat livers and the influence of estradiol-treatment or castration on the metabolism in male rats were investigated. In the metabolism by liver homogenates, the predominant metabolites were hydroxylated testosterones in males, whereas they were androstanedione, androster-one and 5α-dihydrotestosterone in females. Most of the substrate, testosterone, was easily metabolized in females. In the microsomes, the amount of testosterone un-metabolized remaining was high in males, while most of testosterone was metabolized to 5α-androstane-3α, 17β-diol in females. In the supernatant, a large amount of testosterone remained unchanged in females, whereas it was mainly metabolized to 5β-androstane-3α, 17β-diol in males. Castration and estradiol-treatment of male rats induced the female type of metabolic pattern to some extent.