Ca2+ channel blockers belonging to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes (Santos et al., J. Neurochem. 52, S49D, 1989). We now report that (-)-desmethoxyverapamil and flunarizine are the most potent inhibitors of the Ca(2+)-activated ATPase activity of synaptic plasma membranes, decreasing the Vmax by 41% and 37%, respectively, with no significant effects on the Km for Ca2+ (162.7 +/- 14.9 nM free [Ca2+]), while nitrendipine did not affect these parameters. Trifluoperazine was the most potent inhibitor of the Ca(2+)-activated ATPase of synaptic plasma membranes with an IC50 of 8-10 microM. To clarify whether the inhibitory effects of Ca2+ channel blockers and of trifluoperazine on the (Ca2+ + Mg2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125I-calmodulin to the membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer to nitrocellulose and autoradiography. The autoradiograms revealed Ca(2+)-dependent CaM binding proteins of about 140, 70 and 55 kDa. Trifluoperazine (30-40 microM) inhibited by 50-60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca2+ + Mg2+)-ATPase protein. Flunarizine and (-)-desmethoxyverapamil (100 microM) inhibited the 125I-calmodulin binding to the 140 kDa peptides by 100 and 90%, respectively, and they inhibited by 55 and 40%, respectively, the binding of 125I-calmodulin to the peptides in the 70-55 kDa range, whereas nitrendipine did not show any effect. The results suggest that the inhibitory effects of (-)-desmethoxyverapamil and flunarizine, as well as trifluoperazine, on the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.