The relationship between calcium binding and ATPase activity has been investigated for guinea pig cardiac sarcolemma. The concentrations of calcium ions and of ATP were the main factors in determining the amount of calcium bound. With a saturating concentration of ATP, at low calcium concentrations (0.1 mM) more than 50% of the total calcium bound was ATP dependent while at high concentrations of calcium (10 mM) only 20% of the calcium bound was ATP dependent. The ATP-dependent calcium binding process involves one class of calcium binding sites while the non-ATP-dependent calcium binding process involves two classes of binding sites.Inhibitor studies of Ca2+-stimulated MgATPase, MgATPase, and CaATPase activities suggest Ca2+ and Mg2+ are either interacting with different sites on the same enzyme or with different enzymes.