K⁺ channel openers (KCOs) are known to have a wide range of effects by opening the K⁺ channel in plasma membranes of various smooth muscles, cardiac muscle and pancreatic β-cell. In the present study, we investigated the effects of 5 types of KCOs, cromakalim, RP49356, pinacidil, nicorandil and diazoxide on the contractility of isolated rat uterus. All KCOs tested inhibited the uterine contraction induced by 0.2 nM oxytocin in a dose-dependent manner. Individual KCO and its pD₂ values were cromakalim 6.5, RP49356 6.3, pinacidil 5.92, nicorandil 4.43 and diazoxide 4.18. The relaxant effects of KCO were inhibited by glibenclamide (0.3, 1 and 10μM) with pA₂ values of cromakalim 6.91, RP49356 6.59, pinacidil 6.55, nicorandil 5.97 and diazoxide 6.37. In addition, the relaxant effect of cromakalim or pinacidil was antagonised by TEA, a non-selective K⁺ channel blocker, but not by apamin. Contractions induced by low concentration of KCI (< 40 mM) were inhibited by cromakalim (100μM) and nicorandil (300μM), but those evoked by higher concentration (> 40 mM) of KCI were little affected. In ovariectomized rat uterus, cromakalim dose-dependently inhibited oxytocin-induced contraction and glibenclamide (10μM) inhibited the relaxant effect of cromakalim with pD₂ and K_B values of 7.48 and 1.26 × 10^-7M, respectively. In estrogen-primed rat uterus, these values were 6.51 and 1.57 × 10^-7M, respectively, indicating that the cromakalim is less effective on the estrogen-treated uterine smooth muscle. Our results suggest that the KCO-sensitive K⁺ channels participate in the motility of uterine smooth muscle and such channels are, at least in part, under the control of estrogen. In addition, our data Indicate that the type of K⁺ channels activated by KCO is ATP-sensitive K⁺ channels which is blocked by glibenclamide.