Cytochromes P450 3A3/4 are inordinately important P450 enzymes catalyzing the metabolism of a large variety of clinically useful drugs, steroids, and carcinogens. Two monoclonal antibodies, MAb 3-29-9 and MAb 275-1-2, were prepared to human P450 3A4 from mice immunized with baculovirus-expressed human P450 3A4. MAb 3-29-9 was a powerful inhibitor of the enzymatic activity of P450 3A3/4/5. MAb 3-29-9 inhibited the P450 3A3, 3A4, and 3A5 catalyzed metabolism of substrates of divergent molecular weights, e.g., p-nitroanisole, phenanthrene, diazepam, testosterone, taxol, and cyclosporin. However, MAb 3-29-9 did not give a western blot with P450 3A3 or 3A4. MAb 275-1-2 was non-inhibitory but yielded a strong western blot with P450 3A3 and 3A4 but not with 3A5, and thus distinguished between 3A3/4 and 3A5. The two MAbs did not cross-react with human 2E1, 1A2, 2B6, 2C8, and 2C9; rat 2A1, 3A1/2, 4A1, 4A3, and 2B1; and mouse 1A1 and 1A2. MAb 3-29-9 has been used successfully to measure the quantitative contribution of P450 3A3 and 3A4 to the metabolism of the above-designated substrates in human adult liver. MAb 3-29-9 and MAb 275-1-2 are precise and sensitive reagents for P450 3A studies.