Flavin-containing monooxygenases (FMOs) play significant roles in the metabolism of drugs and endogenous or foreign compounds. In this study, the regional distribution of FMO isoforms 1, 3, and 4 was investigated in male Sprague-Dawley rat liver and kidney using immunohistochemistry (IHC). Rabbit polyclonal antibodies to rat FMO1 and FMO4, developed using anti-peptide technology, and commercial anti-human FMO3 antibody were used; specificities of the antibodies were verified using Western blotting, immunoprecipitation, and IHC. In liver, the highest immunoreactivity for FMO1 and FMO3 was detected in the perivenous region, and immunoreactivity decreased in intensity toward the periportal region. In contrast, FMO4 immunoreactivity was detected with the opposite lobular distribution. In the kidney, the highest immunoreactivity for FMO1, -3, and -4 was detected in the distal tubules. FMO1 and FMO4 immunoreactivity was also detected in the proximal tubules with strong staining in the brush borders, whereas less FMO3 immunoreactivity was detected in the proximal tubules. Immunoreactivity for FMO3 and FMO4 was detected in the collecting tubules in the renal medulla and the glomerulus, whereas little FMO1 immunoreactivity was detected in these regions. The FMO1 antibody did not react with human liver or kidney microsomes. However, the FMO4 antibody reacted with male and female mouse and human tissues. These data provided a compelling visual demonstration of the isoform-specific localization patterns of FMO1, -3, and -4 in the rat liver and kidney and the first evidence for expression of FMO4 at the protein level in mouse and human liver and kidney microsomes.