The aim of this study was to investigate the nature of the non-adrenergic non-cholinergic (NANC) inhibitory transmitter of the circular muscle of the porcine ileum. For this purpose, the effects of putative NANC mediators i.e. NO, vasoactive intestinal polypeptide (VIP) and ATP were measured in isolated organ bath experiments (in basal conditions and after incubation with neostigmine 3 x 10[-5] M) and using the microelectrode technique. The NO donor sodium nitroprusside (NaNP) up to 10(-4) M, VIP up to 10(-7) M and ATP up to 10(-4) M failed to cause significant relaxation in the basal state. However, all of them induced marked relaxations when the tissue had been preincubated with neostigmine (3 x 10[-5] M) which was added to increase basal mechanical activity. The resting membrane potential (RMP) was unaffected by NaNP(up to 10(-4) M and VIP up to 10(-7) M whereas ATP (up to 10[-4] M) induced a transient hyperpolarization. The inhibitory junction potentials (IJPs) induced by electrical field stimulation (EFS) were not affected by N omega-nitro-L-arginine (L-NNA) (10[-4] M) whereas suramin, a purinoceptor antagonist, decreased (10[-4] M) or abolished (10[-3] M) the IJPs. Relaxations induced by ATP in neostigmine preincubated tissue were resistant to 10(-6) M tetrodotoxin, an axonal blocker, and inhibited by suramin. Apamin (10[-6] M, a small conductance calcium activated potassium channel blocker, completely abolished the IJP (n=5) and significantly decreased the relaxation induced by ATP (n=5). The present data provide support to the hypothesis that ATP is the NANC inhibitory transmitter in the porcine ileum acting on P2 muscular receptors. Nevertheless, VIP and NaNP do also cause relaxation of preparations preincubated with neostigmine.