1. The effects of different K+ channel blockers were studied on nitric oxide (NO)-mediated non-adrenergic non-cholinergic (NANC) relaxations in the canine ileocolonic junction. 2. The non-selective blockers of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA) and the blocker of large conductance Ca(2+)-activated K+ channels, charybdotoxin, potently enhanced the NANC relaxations induced by low frequency stimulation. The blocker of small conductance Ca(2+)-activated K+ channels, apamin, had no effect on electrically-induced NANC relaxations. 3. NANC nerve-mediated relaxations induced by adenosine 5'-triphosphate (ATP), acetylcholine (ACh) and gamma-aminobutyric acid (GABA) were significantly enhanced by 4-AP and charybdotoxin but not by apamin. TEA significantly enhanced the NANC relaxations in response to GABA and ATP while that in response to ACh was abolished. 4. None of the K+ channel blockers had an effect on the dose-response curve to NO, on the noradrenaline-induced contraction or on the relaxation to nitroglycerine (GTN). 5. From these results we conclude that inhibition of prejunctional K+ channels increases the nitrergic relaxations induced by electrical and chemical receptor stimulation of NANC nerves and thus suggests a regulatory role for these prejunctional K+ channels in the release of NO from NANC nerves in the canine ileocolonic junction.