The smooth muscle alpha 1Cb subunit of the L-type calcium channel was expressed alone (CHO alpha 1 cell) or together with the skeletal beta 1 (CHO alpha 1 beta 1 cell) subunit or smooth muscle beta 3 (CHO alpha 1 beta 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channel with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (IBa) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO alpha 1 and CHO alpha 1 beta 3 cells. The shift in the steady-state inactivation curve and the increase in channel inactivation were less pronounced in CHO alpha 1 beta 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased IBa by up to 198% in 33% of the CHO alpha 1 beta 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit IBa in 60% of the CHO alpha 1 beta 3 cells. These results suggest that the beta subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel alpha 1 subunit.