Summary of Work: The flavin-containing monooxygenase gene family contains five known members. These drug metabolizing enzymes have wide, overlapping substrate specificities and participate in the oxidative metabolism of numerous drugs, pesticides and other environmental chemicals. Of these enzymes, we have shown that FMO3 is the most important with respect to drug metabolism in human liver. In addition to FMO3, both FMO4 and FMO5 are expressed in human liver. The properties of FMO5 have been established, but those of FMO4 have been difficult to assess because of difficulty in expressing this isoform in E. coli. The interaction of tricyclic antidepressants with the FMO1 has been studied with chimeras constructed from the rabbit and pig FMO1 orthologs and by site-directed mutagenesis. Introduction of pig amino acids at five positions in the rabbit protein completely changes the properties of the rabbit isoform to those of the pig isoform. One of these positions, 433, appears to be critical in determining whether FMO2 is activated or inhibited by the antidepressants. FMO3 has also been shown to interact with chemical modulators with results dependent upon substrate concentrations; activation at high concentrations and inhibition at low concentrations. As with FMO1 the amino acid present in position 430 (433 in FMO1) is found to be critical in the regulation of activity by cheimcal modulators. This finding has been examined with FMO2 and similar results have been obtained. Results obtained with recombinant human FMOs are being examined in human liver microsomal samples.