The binding properties of two major and chemically distinct calcium antagonists, [3H]-nitrendipine and [3H]-diltiazem, were investigated in highly purified rat cardiac sarcolemma. In the case of [3H]-nitrendipine, there appeared a single set of high affinity binding sites with a B max of approximately 0.9 pmol X mg-1 protein and a KD of approximately 0.15 nmol/l. Because of the extremely high value obtained for KD (29 mumol/l), the specificity of [3H]-diltiazem binding seemed questionable. The specific binding of 0.1 nmol/l [3H]-nitrendipine to cardiac sarcolemma was inhibited by nitrendipine and nifedipine (1 mumol/l), only partly inhibited by verapamil (1 mumol/l), and was enhanced by diltiazem (0.1-10 mumol/l). The stimulation of [3H]-nitrendipine binding by diltiazem was associated with an increase in the number of binding sites, Bmax, but with no change in the KD or the Hill coefficient. An enantiomer of diltiazem (1-cis) neither stimulated nor inhibited the [3H]-nitrendipine binding. These results strongly suggest that major prototype calcium antagonists have distinct and variously interacting sites of action in the rat cardiac sarcolemma.