The interaction of 1,4-dihydropyridine derivatives with their receptors on voltage-dependent calcium channels in cardiac membranes was studied to determine if there are basic differences in the binding properties of ligands that cause inhibition or activation of calcium channels. The binding characteristics of 6 pure stereoisomers, (-) and (+)202-791, (-) and (+)Bay k 8644, (-) and (+)PN 200-110, as well as racemic Bay k 8644 and nitrendipine, were compared. Competition studies using the cold ligands and 3 different radiolabelled dihydropyridines, (+)[3H]PN 200-110, (±)[3H]nitrendipine, and (±)[3H]Bay k 8644, showed that, for each combination tested, the labelled dihydropyridine could be displaced by the cold dihydropyridine. The binding reactions were markedly affected by temperature. The Kdvalues for most compounds were significantly higher (5-19 times) at 0° than at 37°C. In contrast, the affinity of (+)PN 200-110 was similar at 0° and 37°C, but slightly higher at 25°C. A thermodynamic analysis indicated that the binding of the two pure isomers that are Ca2+-channel activators (“agonists”), (-)Bay k 8644 and (+)202-791, was driven entirely by enthalpy and was associated with an unfavorable decrease in entropy. This was in marked contrast to the binding of the inhibitors (“antagonists”). The binding of (+)PN 200-110 and nitrendipine at low temperatures was driven largely or entirely by entropy. Other antagonist-binding reactions were driven mainly by enthalpy but were associated with favorable increases in entropy. The affinity of the three radiolabelled ligands for the dihydropyridine receptor differed 100 times and appeared to be due to large differences in dissociation rate constants for each of the ligands. The rates of dissociation of (+)[3H]PN 200-110 and (±)[3H]nitrendipine, but not of (±)[3H]Bay k 8644, were significantly slowed by diltiazem, a calcium-channel inhibitor that binds to another receptor on the calcium channel. The results show that there were marked differences in the binding of the various dihydropyridines and suggest that the energetics of binding of Ca2+-channel activators and inhibitors may be fundamentally different.