Differential distribution of alpha-subunit (S100-alpha) and beta-subunit (S100-beta) of S100 protein in nonnervous tissues was studied by employing the indirect peroxidase-labeled antibody method with monospecific antibodies to human S100-alpha or bovine S100-beta. It became clear that S100-alpha and S100-beta were more widespread than previously reported. In light of this study, together with the previous report describing quantitative differential distribution of S100-alpha and S100-beta (Kato K, Kimura S: Biochim Biophys Acta, 842:146, 1985), S100-alpha was localized at much higher levels in myocardial cells and slow-twitch muscle fibers, at high levels in renal tubules, chondrocytes, follicular cells of thyroid, exocrine cells of salivary, mammary, and eccrine sweat glands, and centroacinar and acinar cells of the pancreas. On the other hand, S100-beta was mainly localized in Schwann cells, chondrocytes, and adipocytes. There were several tissues showing a remarkable differential localization of S100-alpha and S100-beta; heart, skeletal muscle, exocrine tissues such as mammary gland, salivary gland, and pancreas, liver, kidney, testis, epididymidis, and lymphoreticular system. Based on the wide distribution and the characteristic differential localization, the possible biologic significance of S100 proteins and their application for diagnostic pathology were discussed.