Effects of ER-001533 (ER), a newly synthesized vasorelaxant, on the membrane currents were examined in single ventricular cells of guinea pigs. The patch-clamp technique was used in the “whole-cell” and “inside-out” patch configurations. In the whole-cell clamp condition, ER induced a time-independent K+-dominant current, which was inhibited by glibenclamide (1–3 μM), suggesting that ER activated the cardiac ATP-sensitive K+channel (KATP). To elucidate the mechanism of ER-mediated KATPchannel activation, the drug was applied to the inside-out patches before and after channel “run-down.” Since nucleotide diphosphates could induce the channel openings after complete run-down, effects of the drug on the nucleotide diphosphate-induced channel openings were also examined. Before run-down, ER activated the KATPchannel only in the presence of ATP. ER shifted the relation between [ATP]1and the channel activity to the right in a concentration-dependent fashion without a significant alteration of the slope. After channel run-down, ER did not affect the channel openings either in the absence or in the presence of UDP. However, ER could relieve the ATP-γ-S inhibition of the UDP-induced channel openings. Thus, we conclude that ER antagonizes the inhibitory effect of ATP on the KATPchannel in a competitive manner, thereby enhancing the channel openings. (Circulation Research1992;70:1054–1061)