In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.