The 55-kDa high-affinity calcium binding protein (HACBP) was first identified and isolated from skeletal muscle sarcoplasmic reticulum (SR). Using polyclonal antibodies raised against the HACBP isolated from skeletal muscle we have identified this protein in cardiac and smooth muscle as well as in non-muscle cells. Although the 55-kDa protein has a size, properties and localization similar to that of calsequestrin, the two proteins are immunologically distinct. The NH2-terminal sequence of uterine HACBP is also completely different from that of calsequestrin but it is identical to that of rabbit liver calregulin, a recently identified calcium binding protein. Indirect immunofluorescence staining of frozen sections and culture cells from a variety of tissues shows that the 55-kDa protein localizes predominantly to junctional SR and T-tubule areas in skeletal muscle, to SR in smooth and cardiac muscle cells, and to ER in a variety of non-muscle cells. These data show that the protein is present in a wide variety of tissues and suggest that it is a protein common for both sarcoplasmic and endoplasmic reticulum membranes.