Hydrogen sulphide (H2S) has been recently proposed as a transmitter in the brain and peripheral tissues. Its role in the gastrointestinal tract is still unknown despite some data which suggest an involvement mediating smooth muscle relaxation. The aim of this study was to investigate the effect of this gas on intestinal segments from mouse jejunum and colon, and muscular strips from the human and rat colon. In isolated segments of mouse colon and jejunum, bath applied sodium hydrogen sulphide (NaHS) (a H2S donor) caused a concentration-dependent inhibition of spontaneous motor complexes (MCs) (IC50 121 μmol L−1 in the colon and 150 μmol L−1 in the jejunum). This inhibitory effect of NaHS on MCs was (i) unaffected by tetrodotoxin (TTX), capsaicin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate and N-nitro-L-arginine suggesting a non-neural effect and (ii) significantly reduced by apamin 3 μmol L−1. NaHS concentration-dependently inhibited the spontaneous motility in strips from human colon (IC50 261 μmol L−1) and rat colon (IC50 31 μmol L−1). The inhibitory effect of NaHS on colonic strips was (i) unaffected by the neural blocker TTX (1 μmol L−1) with IC50 183 μmol L−1 for the human colon and of 26 μmol L−1 for the rat colon and (ii) significantly reduced by glybenclamide (10 μmol L−1), apamin (3 μmol L−1) and TEA (10 mmol L−1) with IC50 values of 2464, 1307 and 2421 μmol L−1 for human strips, and 80, 167 and 674 μmol L−1 for rat strips respectively. We conclude that H2S strongly inhibits in vitro intestinal and colonic motor patterns. This effect appears to be critically dependent on K channels particularly apamin-sensitive SK channels and glybenclamide-sensitive K (ATP) channels.