(+)-Bufuralol 1'-hydroxylation, a commonly used marker of hepatic CYP2D6 activity, was investigated in human and rhesus monkey intestinal microsomes and compared with that in hepatic microsomes. The cumene hydroperoxide (CuOOH)-mediated metabolism of (+)-bufuralol suggested that at least two enzymes were responsible for bufuralol 1'-hydroxylation in both human and monkey intestinal microsomes. In contrast, the kinetics of the CuOOH-mediated metabolism in human and monkey livers were monophasic. The Km values for the higher affinity component of the intestinal enzyme(s) of both species were similar to, while the corresponding Vmax values were much lower than, those obtained with the livers. Bufuralol metabolism mediated by NADPH exhibited biphasic kinetics and was less efficient than that observed in the presence of CuOOH in both human and monkey intestines, in agreement with the observations in the livers. Inhibition of bufuralol hydroxylase activity in the intestine and liver preparations from the same species by known CYP2D6 inhibitors/substrates was qualitatively similar. Quinidine was the most potent inhibitor of (+)-bufuralol 1'-hydroxylation in all tissues studied. Western immunoblots using anti-CYP2D6 peptide antibody revealed a protein band in human and monkey intestinal microsomes of the same molecular weight as that observed in the liver preparations. The intestinal CYP2D protein content appeared to be much less than that of liver, and correlated with the (+)-bufuralol hydroxylase activity. Immunoinhibition studies indicated significant (up to 50%) inhibition of the CuOOH-mediated (+)-bufuralol metabolism in human and monkey intestines only by anti-CYP2D6, and not by anti-CYP2A6, or anti-CYP2E1. Inhibition of the bufuralol 1'-hydroxylase activity by anti-rat CYP3A1 was only slight (20%) in human, but marked (60-65%) in monkey intestinal microsomes. The hepatic metabolism of (+)-bufuralol in humans and monkeys was only inhibited (75%) by anti-CYP2D6, but not by anti-CYP3A1. Overall, the results suggest that (1) tissue and species differences exist in the catalysis of (+)-bufuralol 1'-hydroxylation, and (2) CYP2D6-related enzymes are partially or primarily responsible for the bufuralol hydroxylase activity in human and monkey intestines or monkey liver.