The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma-aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum.