Studies with a sarcolemma preparation from guinea pig hearts revealed two classes of Ca2+ binding sites: high affinity sites (Km' of 16 muM) and a 10-fold greater number of low affinity sites (Km' of 800 muM). Low affinity Ca2+ binding was inhibited by ruthenium red and verapamil, suggesting that these sites are located on the external side of the membrane. However, clear-cut competition between H+ and Ca2+ could not be demonstrated with isolated sarcolemma.