The calmodulin inhibitors R24571 and trifluoperazine were found to inhibit competitively the binding of [3H]nitrendipine to a 48,000 X g particulate fraction of rat brain with IC50 values of 1.0 and 18.8 microM, respectively. Equilibrium dialysis was used to test the ability of the dihydropyridines nitrendipine, felodipine, and nicardipine to inhibit the binding of [3H]chlorpromazine, [14C]pimozide, and 45Ca2+ to calmodulin. At dihydropyridine concentrations near the limit of solubility (10 microM), the only significant effect in these three binding experiments was a 26% inhibition of [14C]pimozide binding to calmodulin by nicardipine, indicating that the dihydropyridines do not bind to the same site on calmodulin as chlorpromazine, pimozide, or calcium. Equilibrium dialysis was also used to determine the ability of the dihydropyridines to interact directly with calmodulin. [3H] Nitrendipine bound to calmodulin in a calcium-dependent manner; however, this binding was of a low-affinity, unsaturable nature. These results suggest that the dihydropyridine drugs do not interact with calmodulin at concentrations that are pharmacologically significant.