Cytochrome P450 (CYP) 2A6 is an important enzyme catalysing the metabolism of many drugs, procarcinogens and promutagens. Its role in human liver metabolism of coumarin, 4-nitroanisole, 4-nitrophenol and 7-ethoxycoumarin was analysed with an inhibitory monoclonal antibody (MAb) to CYP2A6. MAbs were derived from a panel of 16 hybridomas which yielded positive enzyme-linked immunosorbent assay (ELISA) results or immunoblots against CYP2A6. The hybridomas were selected from more than 500 clones generated by the fusion of myeloma cells with spleen cells of mice immunized with purified baculovirus-expressed human CYP2A6. The MAbs obtained from four of the 16 hybridomas exhibited strong inhibitory activity to CYP2A6-catalysed phenanthrene metabolism. MAb 151-45-4 was positive and highly specific to CYP2A6 as determined by ELISA and immunoblot, and showed no cross-reactivity with recombinant human CYP 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, as tested with ELISA and immunoblot analyses. MAb 151-45-4 specifically inhibited CYP2A6-catalysed metabolism of phenanthrene, 4-nitroanisole, 4-nitrophenol, coumarin and 7-ethoxycoumarin each by 94–99% and did not inhibit their metabolism catalysed by 10 other human CYPs. The potent inhibitory effect of MAb 151-45-4 was used to define the contribution of human CYP2A6 to the metabolism of coumarin, 4-nitroanisole and 7-ethoxycoumarin in seven human liver microsome samples. Coumarin metabolism in all of the seven samples was inhibited by greater than 94% by MAb 151-45-4 which indicates that essentially all microsome mediated coumarin metabolism in human liver is catalysed only by CYP2A6. Inhibition of 4-nitroanisole and 7-ethoxycoumarin metabolism by anti 2A6 MAb ranged from 22–65% and 8–24%, respectively. The degree of inhibition defines the contribution of CYP2A6 activity to the 4-nitroanisole and 7-ethoxycoumarin metabolism in human liver and the range reflects the variability among samples. The inhibitory antibody to CYP2E1 was used to determine its role in 4-nitroanisole and 7-ethoxycoumarin metabolism in seven human liver samples. The addition of both MAbs to CYP2A6 and 2E1 to the microsome samples defined combinatorially the relative role of CYP2A6 and 2E1 in the metabolism of 4-nitroanisole and7-ethoxycoumarin.