Abstract Sarcolemma vesicles isolated from rabbit ventricular muscle showed a high degree of purity when assessed in terms of calcium binding and marker enzyme activity. Calcium binding to the sarcolemma increased as the free calcium concentration of the medium increased. ATP-independent (passive) and ATP-dependent (active) calcium binding activities were observed. The active binding was associated with an increase in the calcium stimulated ATPase activity of the preparation. A Lineweaver-Burke plot of the ATP-dependent calcium binding data was linear over the whole range of calcium concentrations (20–200 μM) investigated.