The effects of cytochrome P450 (P450, CYP) ligands and permeabilization of microsomes on 3-hydroxybenzo(a)pyrene [3-OH-B(a)P] glucuronidation mediated by rat hepatic microsomes were studied. While the UDP-glucuronosyltransferase (UGT) activity with non-permeabilized microsomes from 3-methylcholanthrene (MC)-treated rats was markedly reduced by α-naphthoflavone (NF), this inhibitor had hardly any effect when permeabilized microsomes were used in which the inhibitor was expected to have easy access to UGT. Kinetic analysis indicated that the inhibitory effect of α-NF is competitive. These results suggest that a UGT isoform(s) involved in 3-OH-B(a)P glucuronidation is interfered by a CYP1A inhibitor via a mechanism dependent on the intact nature of microsomal membranes in MC-treated rats. It is likely that P450 functions as a substrate transporter for some isoforms of UGT via possible interactions between UGT and P450.