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, the one with the most relevance to humans appears to be FMO3, the major isoform present in adult liver. We have cloned human FMO3 and human FMO5, which is also present in adult liver, and compared their properties. For the metabolism of model drug substrates, FMO3 is far more efficient than FMO5. Based on immunoquantitation with monospecific antibodies, FMO3 and FMO5 are present in human hepatic microsomal preparations in fairly equivalent concentrations. However, kinetic analysis indicates that virtually all of the FMO-mediated drug metabolism in human liver is carried out by FMO3. Of the isoforms of FMO identified, FMO4 is the least understood. This isoform differs from the others in that its derived sequence contains a C-terminal extension of 24 amino acids. Attempts to express FMO4 in E. coli, yeast and COS-1 cells have, until recently, proven unsuccessful. This problem has been overcome by altering the position of the stop codon of the human cDNA and eliminating the C-terminal extension. The modified cDNA is highly expressed in E. coli. Expression of the truncated FMO4 is blocked by inclusion of the 3'extension even when the early stop codon is maintained. However, addition of the FMO4 3'extension to the cDNA for FMO3 has no effect on expression of FMO3. Examination of E. coli transcripts indicates that the lack of expression associated with the FMO4 3'extension is a function of translation, not transcription. The negative effect of the FMO4 3'extension is also observed in an in vitro translation system. The interaction of tricyclic antidepressants with the FMO has been studied with chimeras constructed from the rabbit and pig FMO1 orthologs. Areas of the proteins associated with activation and inhibition have been identified and specific amino acid residues are being investigated by site-directed mutagenesis.