Hydrogen sulphide (H2S) is an endogenous mediator producing a potent relaxation response in vascular and non-vascular smooth muscles. While ATP-sensitive potassium channels are mainly involved in this relaxant effect in vascular smooth muscle, the mechanism in other smooth muscles has not been revealed yet. In the present study, we investigated how H2S relaxes non-vascular smooth muscle by using intact and S-escin permeabilized guinea-pig taenia caecum. In intact tissues, concentration-dependent relaxation response to H2S donor NaHS in carbachol-precontracted preparations did not change in the presence of a K-ATP channel blocker glibenclamide, adenylate cyclase inhibitor SQ-22536, guanylate cyclase inhibitor ODQ, protein kinase A inhibitor KT-5720, protein kinase C inhibitor H-7, tetrodotoxin, apamin/charybdotoxin, NOS inhibitor L-NAME and cyclooxygenase inhibitor indomethacin. We then studied how H2S affected carbachol- or Ca2+-induced contractions in permeabilized tissues. When Ca2+ was clamped to a constant value (pCa6), a further contraction could be elicited by carbachol that was decreased by NaHS. This decrease in contraction was reversed by catalase but not by superoxide dismutase or N-acetyl cysteine. The sarcoplasmic reticulum Ca2+-ATPase pump inhibitor, cyclopiazonic acid, also decreased the carbachol-induced contraction that was further inhibited by NaHS. Mitochondrial proton pump inhibitor carbonyl cyanide p-trifluromethoxyphenylhydrazone also decreased the carbachol-induced contraction but this was not additionally changed by NaHS. The carbachol-induced Ca2+ sensitization, calcium concentration-response curves, IP3- and caffeine-induced contractions were not affected by NaHS. In conclusion, we propose that hydrogen peroxide and mitochondria may have a role in H2S-induced relaxation response in taenia caecum. (C) 2011 Elsevier B.V. All rights reserved.