Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two “intrinsic glycoproteins,” while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.