In membranes from the rat cerebral cortex, the benzothiazepine [3H]diltiazem and the dihydropyridine [3H]nitrendipine label distinct but allosterically interacting calcium channel antagonist recognition sites. In the present study we evaluated the relationship between Ca++ and calcium channel antagonists binding sites within the voltage-dependent Ca++ channel. [3H]Nitrendipine binding to the rat cerebral cortex at 37 degrees C is not inhibited by Ca++, studied as CaCl2, in concentrations up to 10 mM. In contrast, [3H]diltiazem binding under these conditions is inhibited by Ca++ with an IC50 of 0.31 mM. The inhibition of diltiazem binding by Ca++ is reflected in an increase in the EC50 of diltiazem for enhancement of [3H]nitrendipine binding at 37 degrees C but not in the magnitude of its maximal heterotropic positive cooperative effect. Similarly, Ca++ increases the IC50 of verapamil for inhibition of [3H]nitrendipine binding at 37 degrees C without affecting the magnitude of its heterotropic negative cooperative effect. Schild analyses of these data indicate that Ca++ is an essentially competitive antagonist of the allosteric effects of diltiazem (KB = 0.32 mM) and verapamil (KB = 0.33 mM). At 0 degrees C, Ca++ is a negative allosteric inhibitor of the [3H]nitrendipine binding site (IC50 = 0.1 mM) and selectively increases the IC50 of diltiazem and verapamil for the inhibition of [3H]nitrendipine binding to membranes from the rat cerebral cortex. It may thus be suggested that the diltiazem and verapamil recognition sites are closely linked or identical with Ca++ binding site within the slow voltage-dependent Ca++ channel.