Cytochrome P-450 reductase and cytochrome P-450 fractions have been separated and partially purified from colonic mucosal microsomes of rat pretreated with phenobarbital or beta-naphthoflavone. Colonic cytochrome P-450 reductase has a molecular weight of 76,000. The Km values of colonic cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, ferricyanide, and dichlorophenolindophenol and the electron donor NADPH are 6, 50, 11 and 11 microM, respectively. Immunochemical techniques identified the presence of beta-naphthoflavone Forms 1, 4 and 5 after beta-naphthoflavone treatment but beta-naphthoflavone Forms 1 and 4 and phenobarbital Form 1 after phenobarbital treatment.