The neurotransmitter of the non-adrenergic non-cholinergic inhibitory innervation of the stomach is still unknown. We studied the effect of a series of neurotransmitter candidates, ATP, [Leu]enkephalin and [Met]enkephalin, somatostatin, neurotensin and VIP, in the rat gastric fundus and compared these effects with the response to electrical stimulation of the non-adrenergic non-cholinergic inhibitory neurons. Rats of both sexes were treated with reserpine (5 mg . kg-1 intraperitoneally) 24 h before killing. Longitudinal muscle strips of the gastric fundus were prepared and mounted between parallel platinum electrodes in Krebs solution containing atropine 10(-6) M and serotonin 3.10(-6) M. A maximal relaxatory response was obtained on transmural stimulation of the strips at supramaximal voltage, 1 msec and 5 Hz. ATP (10(-6)-10(-3) M) elicited a biphasic response, a small relaxation followed by a contraction. The maximal relaxatory response induced by ATP was much lower than that induced by transmural stimulation during 45 sec (37.3% versus 166.2%, where 100% is the maximal contractile response to ATP, n = 17). Desensitization to ATP did not influence the relaxation induced by transmural stimulation. [Met]enkephalin, [Leu]enkephalin and naloxone did not change the tone of the strips or the amplitude of the electrically induced relaxation. Somatostatin had no influence while neurotensin induced a concentration-dependent contraction from 10(-9) M or 10(-8) M on. VIP (10(-10)-3.10(-8) M) induced a concentration-dependent relaxation. The maximal relaxation induced by VIP was 120.8% of that induced by transmural stimulation (n = 16). The relaxation induced by VIP 10(-8) M, left in contact with the tissue for 10 min, was comparable to that induced by transmural stimulation during 10 min, except for a lag time of more than 10 sec after the addition of VIP. The relaxation induced by VIP was not influenced by tetrodotoxin, phentolamine or propranolol. The peptidase trypsin (10(-6) M) antagonized the relaxation by exogenously added VIP but did not influence the electrically induced relaxation. The results obtained in this study show that, of the substances tested, only VIP mimics the relaxation induced by stimulation of the inhibitory non-adrenergic non-cholinergic neurons in the rat gastric fundus; VIP therefore seems a reasonable candidate as neurotransmitter of these neurons.