Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites ( =0.30 ± 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there arealso dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing lig and, and half-maximal effects occurred at approximately 10 µ for the agonist (±)-BayK8644 and for the antagonists nifedipine, (±)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereo specific since (-)-PN200-110 (1-200 µ ) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-0-(3-thiotriphosphate) (GTPγS) and guanosine 5,-0-(2-thiodiphosphate)(GDPβS) on binding parameters. At a concentration of 10 µ , neither GTPγS nor GDPβS significantly affected the binding of dihydropyridines to their high-affinity sites. GTPγS did, however, increase the ability of (±)-Bay K8644, but not of (i)-nitrendipine, to accelerate the rate of dissociation of tightly bound(+)-[3H]PN200-l 10. GDPβS did not affect the dose dependence of either the agonist or the antagonist.These results suggest that skeletal muscle dihydropyridine receptors have low-affinity binding sites that maybe involved in the regulation of calcium channel function and that activation of a guanine nucleotide bindingprotein may modulate the binding of agonists but not of antagonists to these sites.