The presence of a diltiazem recognition site within the macromolecular complex of the calcium channel in the brain had been hypothesized on the basis of binding studies with [3H]dihydropyridine calcium channel antagonists. In the present study, we therefore characterized [3H]diltiazem binding sites in the rat cerebral cortex. Saturable high affinity (Kd = 50-170 nM) [3H]diltiazem binding to the rat cerebral cortex was stereospecifically inhibited by the enantiomers of diltiazem according to their activity as calcium channel antagonists and modulators of [3H]dihydropyridine binding. An association between the [3H]diltiazem binding site and the calcium channel was further corroborated by the effects of chemically heterogeneous calcium channel antagonists on [3H]diltiazem binding. Dihydropyridines appeared to allosterically affect [3H]diltiazem binding according to their pharmacological effects; e.g. at 37 degrees C nitrendipine enhanced whereas the calcium agonist Bay K 8644 failed to affect [3H]diltiazem binding at concentrations fully inhibiting [3H]nitrendipine binding. The effect of nitrendipine may, at least in part, be explained by an increase in the affinity of [3H]diltiazem.