Hydrogen sulfide has been recently suggested to act as a physiological mediator in the brain and other organs. Here, we used three approaches to study the so far unknown role of hydrogen sulfide as a neuromodulator in the gut. First, immunohistochemistry revealed the presence of the two main hydrogen sulfide producing enzymes cystathionine-g lyase and cystathionine-b synthase in over 90% of neurones in the guinea-pig and human submucous plexus and in guinea-pig myenteric plexus. Second, Ussing chamber studies showed that application of the hydrogen sulfide donor NaHS (0.1–2.5mM) resulted in a significant increase of ion secretion in guinea-pig ileum and colon and in human colon. This increase was tetrodotoxin-sensitive (0.5mM) and inhibited by capsaicin desensitization (10mM) and by the vanilloid receptor antagonist capsazepine (10mM). Third, neuroimaging with potentiometric dyes revealed that NaHS (0.5mM) evoked action potential discharge in 23% of guinea-pig and 34% of human submucous plexus neurons. Action potential frequency was strongly reduced by capsaicin desensitization and capsazepine. The results indicate that hydrogen sulfide can be synthesized in enteric neurons and may function as a pro-secretory mediator and a novel signalling molecule acting via sensory afferent fibres.