The development of sex differences in ethylmorphine N-demethylation and several components of the reaction chain were studied in hepatic microsomes from mice of the CPB-SE strain between 3–11 weeks of age. Sex-specific changes were observed in demethylation rate, type 1 spectral interaction, cytochrome P450 content, and ethylmorphine-induced stimulation of NADPH-cytochrome P450 reductase activity. These changes occurred mainly between weeks 3–7 and were confined to females. It is concluded that the development of the cytochrome P450 system is repressed by androgen during sexual maturation. The kinetic constants of demethylation developed differently from ethylmorphine binding constants. Changes in demethylase were mainly restricted to K m , whereas the changes in type 1 binding only involved the maximum spectral change. In combination with differences observed between the developmental patterns of demethylation rate and cytochrome P450 reductase activities, this demonstrated that the reduction of cytochrome P450-substrate complex is not rate-limiting in ethylmorphine demethylation. The type 1 spectral change was correlated with the amount of cytochrome P450 only when a large portion of the cytochrome was considered inactive in ethylmorphine binding. It is suggested that immature animals possess a low basal level of ethylmorphine binding type 1 sites, which is elevated selectively in females during sexual maturation.