Binding studies were performed to characterize the inhibition by amiloride, 3,4-dichlorobenzamil and quinacrine of specific binding of (+)-[3H]isradipine to L-type voltage-operated calcium ion channels in rat cardiac membranes at 37 degrees C with and without 10(-3) M calcium added. By analysis of saturation, inhibition and dissociation curves we find that without the addition of calcium, amiloride (constant of inhibitor producing 50% inhibition (K0.5) = 6.9 x 10(-4) M, Hill coefficient (nH) = 1.99, k-1 increased) and 3,4-dichlorobenzamil (K0.5 = 7.7 x 10(-7) M, nH = 1.13, k-1 increased) inhibit (+)-[3H]isradipine binding by complex, allosteric interactions, suggesting positive cooperativity between sites for the inhibitors. Quinacrine (K0.5 = 6.7 x 10(-6) M, nH = 0.84, k-1 increased) inhibits the binding allosterically by an action compatible with binding to one site. Addition of 10(-3) M calcium affected the inhibition by amiloride (K0.5 = 1.02 x 10(-3) M, nH = 1.41) and quinacrine (K0.5 = 3.3 x 10(-5) M, nH = 0.65). With calcium added the mechanisms of inhibitions were complex, allosteric, and could be explained by positive cooperativity between sites for amiloride and negative cooperativity between sites for guinacrine. We conclude that calcium addition modulates the inhibitions by amiloride and quinacrine by increasing the inhibition constants and changing the cooperativity.