BACKGROUND: Inhibitory neurotransmission in the human intestine is poorly understood. This study was undertaken to determine the role of nitric oxide (NO), adenosine triphosphate (ATP), and vasoactive intestinal polypeptide (VIP) in inhibitory neurotransmission in human jejunal circular muscle strips. METHODS: In vitro response of precontracted (10(-5) M substance P) normal human jejunal muscle strips to electric field stimulation (EFS) under adrenergic and cholinergic receptor blockade was evaluated. Selective neural blockade was obtained by the NO synthase inhibitor l-NG-nitroarginine methyl ester (l-NAME, 10(-3) M), VIP receptor antagonist (4-Cl-d-Phe6Leu17-VIP, 10(-7) M), P2 purinergic receptor blocker suramin (3 x 10(14) M), or the calcium-dependent potassium channel blocker apamin (10(-6) M). Force generated in response to EFS was quantitated and analyzed statistically. RESULTS: Exogenous NO and ATP dose-dependently inhibited contractile activity and relaxed muscle strips with a concentration yielding a 50% effect (ED50) of 4.5 +/- 2.9 x 10(-6) M and 3.3 +/- 1.3 x 10(-4) M, respectively. EFS resulted in relaxation of precontracted muscle strips in all groups. When compared with controls, relaxation was decreased but not abolished by l-NAME (-0.12 +/- 0.03 vs -0.33 +/- 0. 05, -0.07 +/- 0.03 vs -0.34 +/- 0.05, and 0.04 +/- 0.03 vs -0.30 +/- 0.04 at 2, 5, and 10 Hz, respectively, P < 0.011). d-NAME (inactive stereoisomer of l-NAME), 4-Cl-d-Phe6Leu17-VIP, suramin, and apamin did not alter EFS-induced relaxation. CONCLUSIONS: Inhibition of NO synthesis by l-NAME reduced the inhibitory response to EFS, whereas blocking ATP and VIP receptors or other effector pathways had no effect. Our findings indicate that although NO plays a predominant role in inhibitory neurotransmission in human jejunal circular muscle, another neurotransmitter(s) appears to be involved as well. These data may impact on understanding mechanisms of disorders of gut dysmotility.