Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.