Introduction: Hydrogen sulfide (H₂S) is now considered a gasotransmitter in the mammalian body. In the arterial smooth muscle, H₂S activates ATP-sensitive K⁺ (K⁺_ATP) channels, leading to membrane hyperpolarizaion followed by relaxation. However, the H₂S-induced vasorelaxation also involves K⁺_ATP channel-independent mechanisms. In the present study, we examined and characterized relaxant activity of H₂S in the gastrointestinal and bronchial smooth muscle preparations from guinea-pig, rats and mice. Methods: Male Hartley guinea-pigs, ddY mice and Wistar rats were sacrificed by abdominal exsanguinations under urethane (1.5 g/kg, i.p.) anesthesia. The ileum, sphincter of Oddi and taneia coli from guinea-pigs, the main bronchi from mice and guinea-pigs, and the colon from rats were used for isometric tension recordings. Effects of cumulatively applied NaHS, a donor of H₂S, were observed in the smooth muscle precontracted with carbachol (CCh) at 1 and 10 µM for bronchial and gastrointestinal preparations, respectively. Results: NaHS in a range of 0.01-3 mM induced concentration-dependent relaxation in all preparations precontracted with CCh. The maximal relaxation caused by NaHS was 70% or more of CCh-induced precontraction in most preparations, except for the guinea-pig bronchi in which less than 20% relaxation was induced by NaHS. In the gastrointestinal preparations, EC₅₀ values (mM) for NaHS were 0.11 in the ileum, 0.20 in the taneia coli, 0.17 and 0.09 in the terminal cavity and ampulla, respectively, of sphincter of Oddi from guinea-pigs, and 0.43 and 0.27 in the distal and proximal colon, respectively, from rats. Glibenclamide, an inhibitor of K⁺_ATP channels, at 10 µM did not affect either EC₅₀ values or the maximal effect for NaHS in any preparations. In the bronchial preparations of mice, the EC₅₀ for the relaxant activity of NaHS was 0.83 mM, and was not affected by glibenclamide, antagonists of NK₁ and NK₂ receptors, and inhibitors of soluble guanylyl cyclase, COX-1 and COX-2. Na₂SO₃, a metabolite of H₂S in the mammalian body, did not induce relaxation in a range of 0.01-1 mM in the sphincter of Oddi of guinea-pigs and bronchus of mice. Conclusions: In the gastrointestinal and bronchial smooth muscle preparations used in the present study, NaHS appears to cause relaxation, the activity being similar among distinct tissues and species, except for the guinea-pig bronchus that poorly responds to NaHS. The NaHS-induced relaxation in any preparations is considered independent of K⁺_ATP channels. Further, it is also clear that the relaxant effect of NaHS in mouse bronchus does not involve endogenous neurokinins, nitric oxide and prostaglandins.