Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c mice immunized with liver microsomal cytochrome P-450 purified from rats treated with 3-methylcholanthrene (MC-P-450). Thirty-seven independent hybrid clones among 66 tested produced monoclonal antibodies to the MC-P-450 as measured by radioimmunoassay. More than 10 of the monoclonal antibodies formed were positive for MC-P-450 with respect to protein binding measured by radioimmunoassay, precipitation of the enzyme caused by antibody binding and enzyme aggregation, and inhibition of enzymatic activity. Analysis by gel electrophoresis indicated that a single microsomal protein band interacted with the antibody and that this band comigrated with MC-P-450. These monoclonal antibodies interacted with the major form of cytochrome P-450 from β-naphthoflavone-induced rats as well as with MC-P-450 but did not bind, precipitate, or inhibit the activity of the major form of cytochrome P-450 from phenobarbital-treated rats. The monoclonal antibodies inhibited 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylation activity of the purified MC-P-450 with varying degrees, up to 90%, the latter as measured by the aryl hydrocarbon hydroxylase assay for phenol production. Analysis of benzo(a)pyrene metabolism by high-pressure liquid chromatography indicated that the monoclonal antibodies inhibited the enzyme activity of the purified MC-P-450 at all of the positions at which oxidation occurs. The monoclonal antibodies also inhibited both aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase of liver microsomes from 3-methylcholanthrene-treated rats by 70%, indicating that these activities are functions affected by antibody binding to a common or identical antigenic site on cytochrome(s) P-450 which account for 70% of the total activity in these microsomes. Microsomes from control or phenobarbital-treated rats were unaffected, suggesting that their enzyme activity is a function of a cytochrome P-450 other than that sensitive to the MC-P-450 directed antibody and that the latter P-450 is absent in these microsomes. High-pressure liquid chromatographic analysis of antibody inhibition of benzo-(a)pyrene metabolism by microsomes from 3-methylcholanthrene-treated rats showed an inhibition of phenol and diol formation that ranged from 46 to 72%. 1,6-Quinone production was not affected, which suggests that this metabolite was formed by another cytochrome P-450 isozyme or nonenzymatically. The purity, specificity, and potential immortality of their hybridoma cell source will make the monoclonal antibodies extraordinarily useful for the study of substrate and inducer specificity and in the identification and quantitative assay of multiple forms of the cytochrome P-450 and the determination of their content and function in different tissues, species, and individuals.