Cardiac calsequestrin was extracted from canine cardiac sarcoplasmic reticulum vesicles with Nonidet P-40 and purified by precipitation with calcium phosphate followed by fractionation on DEAE-cellulose. It bound 300 nmol of Ca2+/mg of protein and glutamic and aspartic acid constituted approximately 32% of the amino acid residues in the protein. The apparent molecular weight of canine cardiac calsequestrin was 55,000 when measured in alkaline sodium dodecyl sulfate gels and 44,000 when measured in neutral sodium dodecyl sulfate gels. Cardiac calsequestrin, like skeletal muscle calsequestrin, stains blue with “Stains-all” and is therefore the 55,000 dalton protein which Jones and Cala ((1981) J. Biol. Chem 256, 11809-11818) identified as a component of ryanodine-sensitive cardiac sarcoplasmic reticulum. Unlike skeletal muscle calsequestrin, cardiac calsequestrin was sensitive to endo-S-Nacetylglucosaminidase H, indicating that it contained a “high mannose” oligosaccharide. Cardiac calsequestrin cross-reacted with an antiserum raised against skeletal muscle calsequestrin. Indirect immunofluorescent staining of rat cardiac muscle using the skeletal muscle calsequestrin antiserum revealed a polygonal pattern in transverse sections and I-band staining in longitudinal sections. A comparison between the localization of the (Ca2++ M&+) ATPase and calsequestrin suggested that cardiac calsequestrin was confined to the regions of cardiac muscle cells where the sarcoplasmic reticulum was localized. A 53,000 dalton, intrinsic glycoprotein was also identified in cardiac sarcoplasmic reticulum by endo-P-Nacetylglucosaminidase H digestion,‘251-concanavalin A binding, and indirect antibody staining with skeletal muscle glycoprotein antiserum and ‘251-protein A. Its molecular weight of 53,000 was reduced to 49,000 by endo-8-N-acetylglucosaminidase H digestion. Cardiac sarcoplasmic reticulum also contained a Stains-all blue staining, 130,000 dalton glycoprotein, which was similar to the 160,000 dalton glycoprotein of skeletal muscle sarcoplasmic reticulum.