The relationship between the changes in the amount of the components of the liver microsomal electron transport systems and drug hydroxylase activities on administration of p-nitroanisole was investigated. The content of cytochrome P-450 in the p-nitroanisole-treated rats was not significantly different from that in the controls during the treatment. The cytochrome b5 content increased 2.2-fold over that of the controls after 14 days of treatment. Demethylation activities per mg microsomal protein of p-nitroanisole, aminopyrine, and benzphetamine were enhanced 2.7-, 2.4-, and 2.8-fold, respectively, but aniline hydroxylase activity was not enhanced. Similar results were obtained with microsomes from diisopropyl 1,3-dithiol-2-ylidenemalonate (NKK-105)-treated rats. The time-course study of changes in the amount of the components of drug hydroxylase systems suggested that the increase in cytochrome b5 content enhanced the demethylation activities. In the reconstituted system containing cytochrome P-450 partially purified from p-nitroanisole- or NKK-105-treated rats, cytochrome b5 was required for the maximal activities of the demethylation reactions, but did not participate in aniline hydroxylation. These findings showed good correlation between the change of cytochrome b5 content and the demethylation activities and suggested that the increase in cytochrome b5 content might enhance the demethylation activity by stimulating supply of a second electron to cytochrome P-450.