The verapamil-like calcium channel modulator, (-)-[3H]desmethoxyverapamil binds to multiple sites in microsomal membrane preparations from brain and skeletal muscle. In brain the Kd values of the sites are 0.55 +/- 0.1 and 61.8 +/- 18 nM for the high and low affinity sites and the maximum binding values are 0.22 +/- 0.04 and 4.6 +/- 1.0 pmol/mg of protein, respectively. Equilibrium analysis of saturation data in skeletal muscle membranes shows only one site with an affinity of 7.2 +/- 0.8 nM and a maximum binding of 2.96 +/- 0.32 pmol/mg of protein. However, a low affinity site with an estimated Kd of 152 nM is indicated in dissociation kinetic studies. Dihydropyridine calcium channel modulators regulate the binding of desmethoxyverapamil in a temperature-dependent fashion with (+)-PN 200110 decreasing (-)-[3H]desmethoxyverapamil binding more at 0 degrees C than at higher temperatures and, at 37 degrees C, enhancing binding in skeletal muscle. The influence of (+)-desmethoxyverapamil on (+)-[3H]PN 200110 binding is unchanged by temperature variations, whereas interactions of the (-)-enantiomer are altered markedly with more inhibition at 0 degrees C than at higher temperatures and, in skeletal muscle, stimulation of binding at 37 degrees C. Dissociation studies indicate that the two sites labeled by (-)-[3H]-desmethoxyverapamil in skeletal muscle interact in a negative heterotropic cooperative fashion. Dihydropyridines appear to slow the dissociation of ligand from the low affinity site, whereas diltiazem accelerates the dissociation of (-)-[3H]desmethoxyverapamil from the high affinity site. These results suggest that the high and low affinity sites labeled by (-)-[3H]desmethoxyverapamil, respectively, represent the verapamil and diltiazem receptors in brain and skeletal muscle.