The major "male specific" species of cytochrome P-450 (P-450ml) was purified and an antibody against it used to evaluate the involvement of this isozyme in alterations of drug metabolism in senescence. P-450ml exhibited strikingly high imipramine (IM) N-demethylase activity while it showed no IM 2-hydroxylation, which is an alternate pathway of IM metabolism in rat liver microsomes. The antibody to P-450ml inhibited 80% of imipramine N-demethylation in young male rats. In old male rats, which have been shown to have lower IM N-demethylase activity, a 60% inhibition was observed. The inhibitable portion of this activity in old male rats is about one third of that in young rats, but the remaining portion not inhibited by this antibody is almost identical in young and old rats. IM 2-hydroxylation on the other hand was not inhibited by this antibody at all. It also inhibited about 30% of diazepam(DZ) N-demethylation in young rats but showed no inhibition in old rats, resulting in the loss of the age difference in the remaining portion. DZ 3-hydroxylation was not inhibited by this antibody, in spite of the fact that it showed a markedly higher activity in young male than in young female rats with a subsequent reduction in old age in male rats. This study provides the first direct evidence that differences in the amount of the major male specific P-450 isozyme (P-450ml) are responsible for the age- and sex-associated differences in some of the drug metabolizing activities. It also became apparent that P-450ml may not be the only isozyme responsible for these differences.