A rat liver cDNA clone, RFMO1, coding for a flavin-containing monooxygenase (FMO) was isolated. This cDNA clone encoded a protein of 532 amino acids. The deduced amino acid sequence was 84, 82 and 82% identical to those of the pig, human (Form 1) and rabbit (Form 1) liver FMOs, while it was only 52, 50, 54, 56 and 54% identical to the human (Form II), human (Form 2) and rabbit liver FMOs (Form 2) and rabbit and guinea pig lung FMOs. RNA blot analysis showed that rat liver FMO was also expressed in lung and kidney and to a lesser extent in the heart and brain. An expression plasmid, pAMFMO, was constructed and the FMO protein expressed in yeast (AH22). This FMO protein catalyzed thiobenzamide S-oxidation, and NADPH oxidation associated with the S- or N-oxidation of thiourea, N,N-dimethylaniline, trimethylamine, imipramine, chlorpromazine, N,N-dimethylhydrazine, thioacetamide as substrates. The S-oxidation activities of thiobenzamide and thiourea were enhanced by n-octylamine, a known enhancer of FMO, and inhibited by alpha-naphthylthiourea, a known inhibitor of FMO. This is the first report in which FMO with catalytic activities was stably expressed.