Cytochrome P-450 was solubilized from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B and heparin-Sepharose CL-6B columns. Fatty acid omega- and (omega-1)-hydroxylation activity was reconstituted from the partially purified cytochrome P-450 and NADPH-cytochrome c reductase, with phosphatidylethanolamine or phosphatidylcholine. The activity was further stimulated by addition of detergent-solubilized cytochrome b5 from rabbit liver microsomes. Trypsin-solubilized or boiled detergent-solubilized cytochrome b5 had no effect. Among fatty acids tested, caprate, laurate, myristate, and palmitate were the most effective substrates. When caprate and laurate were used as the substrates, the products were the corresponding omega- and (omega-1)-hydroxy fatty acids. The ratio of these products was altered by addition of cytochrome b5. On the other hand, when myristate and palmitate were the substrates, small amounts of unknown polar fatty acids were also formed besides omega- and (omega-1)-hydroxy fatty acids, and the ratio of these products was not affected by addition of cytochrome b5. Benzo(a)pyrene hydroxylation activity was also reconstituted from the same cytochrome P-450 preparation, NADPH-cytochrome c reductase, and phosphatidylserine. However, cytochrome b5 showed only a slight stimulation. The possibility that different cytochrome P-450 species are involved in fatty acid and benzo(a)pyrene hydroxylations is discussed.