Background: Active and significant relaxation of the human gallbladder must be one of the facets of its motility during both the filling and emptying cycle. Conflicting reports about the presence or significance of nitric oxide have been reported in the literature. The aim of this study was to investigate the role of nitric oxide and KATP channels in human gallbladder muscle using isolated strips from human gallbladder. Methods: Full thickness strips were obtained from 56 human gallbladders and suspended under isometric tension in organ baths. The effect of nitric oxide donors and inhibitors on cholecystokinin octapeptide- and carbachol-induced contraction was examined. In separate experiments the effect of the KATP channel activator, cromakalim, and the inhibitor, glibenclamide, were determined. Results: Cromakalim induced a significant relaxation of agonist-induced contraction in human gallbladder in vitro, an effect which was abolished by the KATP channel inhibitor glibenclamide. No evidence of significant nitric oxide involvement in relaxation was observed. Conclusions: This study has demonstrated the presence of KATP channels in human gallbladder for the first time. These are capable of causing significant relaxation in the presence of hormonal and muscarinic agonists and may represent a major pathway for gallbladder relaxation.