The effects of divalent cations and some Ca2+ antagonists on the non-adrenergic inhibitory potential (i.p.) in the duodenal smooth muscle cells of the guinea-pig were investigated intracellularly. The membrane potential was a function of the external Ca2+ (0.25-7.5 mM) and Mg2+ (1.2-24 mM) concentrations. The latency and the time to peak of the i.p. were prolonged by low Ca2+ and excess Mg2+. The amplitude and the rate of hyperpolarization of the i.p. were reduced by low Ca2+ and excess Mg2+. The effects of Mn2+ (5-10 mM) on the i.p. were similar to those by excess Mg2+ except for the small depolarization. The i.p. evoked in the Ca2+-free solution was considerably restored by the addition of Ba2+ (1.25 mM) and Sr2+ (2 mM). The actions of Ba2+ and Sr2+ on the i.p. were inhibited by verapamil (10(-4)g/ml). Verapamil (2 X 10(-5)-2 X 10(-4)g/ml) and gentamicin (10(-3)g/ml) reduced the amplitude and the rate of hyperpolarization of the i.p. The latency and the time to peak of the i.p. were prolonged by verapamil but not by gentamicin. In nifedipine (10(-4)g/ml), the two-peaked i.p. was evoked by a single stimulus. This potential was similar to that evoked by a paired pulse in normal solution. The results obtained suggest that the released of the non-adrenergic inhibitory substance requires Ca2+ which moves into the non-adrenergic inhibitory neurons through the Ca2+ conductance pathway.