S100 protein, an acidic and calcium‐binding protein, was believed to be localized in the nervous tissue, but recently it has been reported to be mainly present in the cardiac and the skeletal muscles of various mammals in the αα form (S100a₀) at much higher levels than the nervous tissues. We isolated here S100 protein from human cardiac muscles. The isolated cardiac muscle S100 protein showed a single band on electrophoresis at the same position as that of human skeletal muscle S100a₀. The amino acid composition of the purified S100 protein was quite similar to that of human skeletal muscle S100a₀ or bovine brain S100a₀. The immunohistochemical study by use of antibodies monospecific to the α subunit of S100 protein (S100‐α) revealed that S100‐α was strongly labeled in human myocardial cells, whereas the β subunit of S100 protein (S100‐β) was not detected in the cells. These results suggest that a predominant form of S100 protein in human myocardial cells is not S100a (αβ) or S100b (ββ), but S100a₀ (αα). In order to determine the ultrastructural localization of S100a₀ in mouse cardiac muscle, the direct peroxidase‐labeled antibody method was employed. S100a₀ was mainly localized in the polysomes in the interfibrillar spaces, the fine filamentous structure of the Z line and fascia adherens of the intercalated disc and in the lumen of junctional sarcoplasmic reticulum.