To clarify the characteristics of KC-399, a newly synthesized potassium channel opener, we investigated the effects of KC-399 and lemakalim on the contractions induced by norepinephrine (NE 1 μM) and K+ (30 and 90 mM) and on 86Rb efflux in rat thoracic aorta. KC-399 (0.01–10 nM) and lemakalim (0.001–10 μM) induced relaxation in aortic rings precontracted with 30 mM K+ or NE, but not with 90 mM K+. The vasorelaxant effect of KC-399 was almost 500 times more potent than that of lemakalim. The vasorelaxation with KC-399 developed more slowly and was more resistant to washout than that induced by lemakalim. Glibenclamide (0.1–1 μM), a blocker of ATP-sensitive K-channels, produced concentration-dependent inhibition of the relaxant action of KC-399 in aorta treated with 30 mM K+ KC-399 (3–100 nM) and lemakalim (0.3–10 μM) stimulated 86Rb efflux in rat aorta; the potency of KC-399 was $100 times greater than that of lemakalim. The effects of KC-399 on 86Rb efflux persisted after an 18-min washout period, but those of lemakalim did not. The stimulatory effects of KC-399 (10 and 30 nM) and lemakalim (1 and 3 μM) on 86Rb efflux were also significantly reduced by glibenclamide (1 μM). These results suggest that KC-399 is a potent and long-lasting vasodilator in vitro and that opening of the ATP-sensitive K+ channel may be involved in its mechanism of action.