Abstract Aims Hydrogen sulphide (H2 S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about H2 S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2 S synthesis throughout the gastrointestinal tract. Methods H2 S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2 S synthesis, cystathionine-γ-lyase and cystathionine-β-synthase, were examined. Cystathionine-γ-lyase and cystathionine-β-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-γ-lyase and cystathionine-β-synthase expression in biopsies of human colon was also examined. Results H2 S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-γ-lyase and cystathionine-β-synthase expression. The efficacy of cystathionine-β-synthase and cystathionine-γ-lyase inhibitors to reduce H2 S synthesis in these tissues was also variable. Cystathionine-β-synthase is the predominant source of H2 S synthesis in the colon of rodents. Cystathionine-γ-lyase and cystathionine-β-synthase were also expressed in healthy human colon biopsies. Conclusions The capacity for H2 S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2 S and the development of more selective inhibitors are suggested.