Abstract: When the concentrations of α-S100 (α subunit of S100 protein) and β-S100 (β subunit) proteins in various tissues of human and rat were determined by the immunoassay method, immunoreactive β-S100 was present at high levels in the CNS, adipose tissue, and cartilaginous tissue. In contrast, the α-S100 was found in the heart and skeletal muscles at concentrations much higher than in the CNS. The concentration of α-S100 protein was also high in the heart and skeletal muscles of bovine, porcine, canine, and mouse. Since β-S100 protein levels in those tissues were low, it was suggested that S100 protein in the muscle tissues is present mainly as the αα form (S100a0 protein). To confirm the above findings, immunoreactive α-S100 protein was purified from human pectoral muscle by employing column chromatographies with butyl-Sepharose, diethylaminoethyl (DEAE)-Sepharose, Sephadex G-75, and finally with an anion-exchange Mono Q column in a HPLC system. The elution profile of α-S100 protein from the Mono Q column suggested some heterogeneity of the final preparation. However, each of these fractions traveled with a single band at a position similar to that of bovine S100a0 protein on slab-gel electrophoresis. The amino acid composition of the final preparation was very similar to the composition of bovine S100a0 protein. The purified α-S100 protein was eluted from a gel-filtration column (Superose 12) in the same fraction as bovine S100a0 protein. These results indicate that the α-S100 protein purified from human pectoral muscle is S100a0 protein, and that S100a0 protein is present universally in the heart and skeletal muscles in a variety of animals at much higher levels than in the brain.