Scorpion venom (Leiurus quinquestriatus), a substance that evokes neurotransmitter release by depolarizing neurons, was used to activate enteric neurons in short-circuited guinea pig ileum. Scorpion venom increased transmural potential difference and short-circuit current, and this response was similar to the increase that occurred after electrical stimulation of enteric neurons. The stimulus- or venom-evoked response in short-circuit current was abolished by tetrodotoxin. Atropine reduced by 47% the increments in short-circuit current produced by either electrical stimulation or venom. Scorpion venom increased active chloride secretion in short-circuited guinea pig ileal mucosa but had no significant effect on active sodium absorption, residual flux, or total tissue conductance. No morphological changes in transmission electron micrographs of ileal mucosa treated with scorpion venom were evident compared with controls. Alanine caused an increase in short-circuit current in venom-treated tissue that was similar to control values. These results show that scorpion venom mimics the mucosal effects of electrical activation of enteric neurons. These results suggest that a significant component of both scorpion venom action and the response to electrical field stimulation is mediated by neural release of acetylcholine, which activates epithelial muscarinic receptors.