BACKGROUND Effects of hydrogen sulfide (H(2)S), a third gasotransmitter of the gut, are not well understood. The aim of this study was to determine effects/mechanisms of H(2)S action on contractile function in rat jejunal muscle. METHODS Transmural strips of longitudinal muscle were evaluated. Response to sodium hydrosulfide (NaHS, H(2)S donor; 10(-5)-10(-3)M) was studied on spontaneous contractile activity and after precontraction (bethanechol, 3 × 10(-6)M). Atropine, propranolol, phentolamine, tetrodotoxin, capsaicin, L-N(G)-nitro arginine (L-NNA), and glibenclamide were used to determine mechanisms. L-cysteine (10(-4)-10(-2)M; substrate for H(2)S production) and aminooxyacetic acid and DL-propargylglycine (inhibitors of enzymes generating H(2)S endogenously) were used to study endogenous production. Aminooxyacetic acid, DL-propargylglycine, L-NNA, and vasoactive intestinal polypeptide (VIP) antagonist [D-p-Cl-Phe(6),Leu(17)]-VIP were used to study H(2)S release during electrical field stimulation (EFS) and interaction with VIP and nitric oxide. Immunohistofluorescence of jejunal whole mounts was performed for endogenous H(2)S-producing enzymes. RESULTS Cystathionine-β-synthase and cystathionine-γ-lyase were expressed only in myenteric plexus. NaHS suppressed spontaneous and stimulated contractile activity (P < 0.01). Glibenclamide prevented some suppression by NaHS (P = 0.01) of stimulated contractile activity but did not prevent suppression of spontaneous contractile activity. Other drugs had no effect on spontaneous contractile activity but increased inhibitory effects of NaHS on spontaneous and stimulated contractile activity (P < 0.05). L-cysteine had no effects on contractile activity. Inhibitors altered basal and stimulated activity suggesting endogenous release of H(2)S. CONCLUSIONS H(2)S presumably suppresses contractile activity in jejunum by direct effects on smooth muscle. Mechanism(s) of inhibition remains unclear, because blocking known neurotransmitters enhanced H(2)S-induced suppression, while blocking adenosine triphosphate (ATP)-sensitive K(+)-channels did not block H(2)S-induced inhibition.