Background. The aim of this study was to investigate the role of omeprazole and lansoprazole, H+-K+ ATPase inhibitors, in gallbladder smooth muscle contractility in vitro. Methods. Gallbladder muscle strips obtained from guinea pigs were mounted in an organ bath. The responses of both precontracted strips and strips under basal tension to omeprazole and lansoprazole were determined. Results. Spontaneous contractile activity was blocked following omeprazole and lansoprazole administration. The agents also caused concentration-dependent relaxation in carbachol- and KCl-precontracted gallbladder muscle strips. Pretreatment with atropine (1 muM), N-w-nitro-L-arginine methyl ester (L-NAME; 30 muM), indomethacin (10 muM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethylammonium chloride (0.5 mM), glibenclamide (1 muM), 4-aminopyridine (0.1 mM), or clotrimazole did not inhibit this relaxation. Gallbladder strips were placed in high-concentrtion potassium (80 mM), calcium-free solution. The contraction produced with the addition of Ca2+ (2.5 mM) was completely relaxed by omeprazole, lansoprazole, and nifedipine separately. Conclusions. These results demonstrate that omeprazole and lansoprazole have potent inhibitory effects on spontaneous contractions and cause dose-dependent relaxation in precontracted gallbladder smooth muscle strips of guinea pig in vitro. This effect could be due to blockade of the calcium channels.