The role of nitric oxide (NO) and ATP in the regulation of nonadrenergic, noncholinergic (NANC) inhibitory transmission in the pylorus remains unclear. In the presence of atropine and guanethidine, electric field stimulation induced NANC relaxations in a frequency-dependent manner (1-20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by N^G-nitro-L-arginine methyl ester (L-NAME; 10⁴ M). P_2X purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 3 × 10⁵ M) and P_2Y purinoceptor antagonist reactive blue 2 (2 × 10⁵ M) had no effect on NANC relaxations. However, the combined administration of L-NAME and PPADS, but not reactive blue 2, evoked greater inhibitory effects on NANC relaxation than that evoked by L-NAME alone. -Chymotrypsin and vasoactive intestinal polypeptide antagonist did not affect NANC relaxations. ATP (10⁵-10³ M) and P_2X purinoceptor agonist ,-methyleneadenosine 5'-triphosphate (10⁷10⁵ M), but not P_2Y purinoceptor agonist 2-methylthioadenosine 5'-triphosphate (10⁷10⁵ M), induced muscle relaxations in a dose-dependent manner, and relaxations were significantly reduced by PPADS and unaffected by TTX. These studies suggest that NO and ATP act in concert to mediate NANC relaxation of the rat pylorus. ATP-induced relaxation appears to be mediated by P_2X purinoceptors located on smooth muscle cells.