The release of endogenous vasoactive intestinal polypeptide (VIP) from enteric nerves of isolated rat ileum and the role of extracellular calcium on the release mechanism have been investigated. Evaluation of simultaneous release of endogenous acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) from enteric nerves was used to establish the reliability of the technique. Electrical field stimulation of the ileal preparation induced an increase in the release of endogenous ACh, ATP and VIP. The evoked, but not the basal, release of these substances was blocked by tetrodotoxin (TTX), indicating that the release was a result of nerve stimulation. However, while increase in release of ACh and ATP during nerve stimulation was suppressed in Ca2+-free medium and by the addition of the Ca2+ channel blocker cadmium, nerve-mediated VIP release was unaffected. Further, while K+-depolarization induced release of ACh and ATP from the ileal preparations, it did not lead to an increase in the release of VIP. These results demonstrate that, unlike ACh and ATP release, release of endogenous VIP from enteric nerves is independent of extracellular calcium. The implications of these results in terms of the mechanism of transmitter release in the gastrointestinal tract are discussed.