In the rat gastric fundus, non-adrenergic, non-cholinergic (NANC) relaxations are mediated by nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and a third, as yet unidentified, neurotransmitter. The possible involvement of adenosine 5'-triphosphate (ATP) in the NANC relaxations was examined using pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (PPADS), apamin and desensitization to alpha,beta-methylene ATP. NANC responses were studied in the absence and presence of N(G)-nitro-L-arginine methyl ester (NAME; 100 microM) and alpha-chymotrypsin (1 u ml(-1)), to inhibit responses to NO and VIP, respectively. PPADS (100 microM), apamin (1 microM) and desensitization to alpha,beta-methylene ATP (10 microM, three additions) all significantly (P<0.05) reduced NANC relaxations to electrical field stimulation (0.5 - 4 Hz, 30 s trains) in longitudinal strips of rat gastric fundus and almost abolished the residual relaxation remaining in the presence of NAME and alpha-chymotrypsin. PPADS had no effect on responses to the NO-donor, sodium nitroprusside (SNP), or VIP. Apamin slightly reduced relaxations to SNP, but did not affect those to VIP, whereas desensitization to alpha,beta-methylene ATP markedly reduced responses to both SNP and VIP. The effects of PPADS and apamin in this study provide strong evidence that the third inhibitory NANC neurotransmitter in the rat gastric fundus is ATP.