The effects of the inorganic Ca2+ channel blockers Cd2+ and La3+ on dihydropyridine (DHP) binding in highly enriched cardiac sarcolemma preparations has been examined. Cd2+ produced an apparent competitive inhibition of DHP binding with a Ki of 60 microM. DHP binding in the presence of La3+ produced nonlinear Scatchard plots when performed in intact membrane vesicle preparations. Evaluation of DHP binding in saponin-permeabilized vesicles or in the presence of the ionophore A23187 yielded linear Scatchard profiles in the presence of La3+. Under these conditions, La3+ produced a mixed-type inhibition, with effects on both Kd and Bmax. These results suggest that La3+ must have access to the interior of sealed vesicles for expression of full inhibitory activity and that La3+ may produce inhibition of DHP binding by interaction with only one surface of the membrane. In order to evaluate the sidedness of the La3+ interaction, membrane preparations consisting of 74% right side out and 26% leaky vesicles were isolated. In the absence of saponin, La3+ decreased maximum DHP binding in this preparation approximately 25%, with no significant change in Kd. When binding was performed in saponin-permeabilized preparations, however, La3+ produced dramatic decreases in both DHP binding affinity and capacity. These results are consistent with the hypothesis that La3+ produces inhibition of DHP binding by interaction with sites accessible only from the cytoplasmic membrane surface. To obtain additional support for this hypothesis. DHP binding was examined in rat ventricular myocytes grown in culture. La3+ and Cd2+, at concentrations in the extracellular buffer that substantially inhibited K+ depolarization-induced 45Ca2+ influx, had little or no effect on DHP binding.