Hydrogen sulfide (H2S) plays an important role in the regulation of gastrointestinal motility and secretion. H2S is synthesized mainly from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS). We showed that smooth muscle cells of stomach and colon of rabbit, mouse and human express CSE, but not CBS. Activation of CSE causes inhibition of Rho kinase activity and muscle contraction and the inhibition was blocked by CSE siRNA and by a selective CSE inhibitor, propargylglycine (PPG).AimTo identify the mechanism of activation of CSE in gastrointestinal smooth muscle cells.MethodsActivation of CSE was analyzed by the measurement of H2S in response to L‐cysteine and NO donor sodium nitroprusside (SNP) in gastric and colonic muscle of rabbit and human. Filter paper disks dipped in zinc acetate were exposed to H2S trapped in N2‐perfused glass vials and the amount of reduced zinc acetate in the presence of H2S was measured spectrophotometrically.ResultsThe basal levels of H2S in gastric and colon muscle from rabbit and human were ~20‐25 nM/mg. L‐Cysteine (10 mM) increased H2S generation up to 50 nM/mg; the effect of L‐cysteine was inhibited by PPG. L‐cysteine‐induced H2S generation was augmented in the presence of SNP and cGMP analog, 8‐bromo cGMP to 125 µM/g and 120 nM/mg, respectively. Augmentation of CSE activity in response to SNP and 8‐br‐cGMP was blocked by the cGMP‐dependent protein kinase (PKG) inhibitor, Rp‐cGMPS, suggesting phosphorylation‐dependent activation.ConclusionWe have identified a mechanism of stimulation of CSE activity in gastric and colonic muscle involving cGMP‐dependent activation of PKG.