Ca 2+ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10 −5 and 1.2 · 10 −3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca 2+ binding were studied. All cations tested inhibited Ca 2+ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na + > K + > Li + ⩾ Cs + and for the divalent and trivalent ions: La 3+ ⩾ Mn 2+ > Sr 2+ ⩾ Ba 2+ > Mg 2+. 1 · 10 −3 M caffeine and 1 · 10 −8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10 −7 M verapamil, acidosis (pH 6.4), 1 −10 −5 M Mn 2+ and 1 · 10 −4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.