A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus-or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60-80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver: p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.