The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site ( K d ) = 265 μM, B max = 65 nmoles mg protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K i values found for divalent metal ions was similar to the K d value for calcium. La +3 produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation ( K i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol ( k i = 115 μM and 108 μM, respectively).