The interaction of the nondihydropyridine calcium channel antagonist Ro 40-5967 with the stably expressed class C alpha 1-subunit of the cardiac L-type calcium channel was investigated and compared with that of verapamil by using the whole cell patch clamp configuration. Both compounds blocked the Ba++ inward current. The IC50 values at a holding potential of -80 or -40 mV were 4.9 and 1.4 microM for Ro 40-5967 and 250 and 15.5 microM for verapamil. Both Ro 40-5967 and verapamil induced a partial tonic block at a holding potential of -80 mV. The block increased with high depolarization rates. Both Ro 40-5967 and verapamil shifted the steady-state inactivation curve by more than 20 mV to hyperpolarized membrane potentials and decreased the inactivation rate constant. The effect of Ro 40-5967, but not that of verapamil, was attenuated by intracellular dialysis with GTP gamma S. The affinity for verapamil was not affected by replacing Ba++ by Ca++, but was increased by the coexpression of the beta 3-subunit. These results indicate that both compounds interact with high affinity with the inactivated channel state, but may interact additionally with the open channel.