1. Changes in isometric tension were recorded from circular muscle strips of rat pyloric sphincter in vitro, in response to electrical field stimulation and exogenously applied muscle relaxants. 2. Concentration-response relationships were studied for relaxation to exogenously applied adenosine 5'-triphosphate (ATP) and two analogues, 2-methylthioATP (2-MeSATP) and alpha,beta-methylene ATP (alpha,beta-MeATP). These drugs evoked concentration-dependent relaxation of rat pyloric sphincter with an order of potency 2-MeSATP > ATP >> alpha,beta-MeATP, indicating the presence of P2y-purinoceptors. The IC50 value of each nucleotide was: 2-MeSATP, 5.0 x 10(-8); ATP, 7.9 x 10(-6) M; alpha,beta-MeATP showed only slight activity at a concentration of 0.1 mM. 3. Frequency-response relationships for relaxations evoked by electrical field stimulation (EFS) were studied in the absence and presence of 10 microM NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide (NO) synthesis) and 20 microM reactive blue 2 (a P2y-purinoceptor antagonist). It was found that these substances significantly reduced the relaxant response of rat pyloric sphincter to EFS by 40% and 50% respectively. In the presence of both L-NAME and reactive blue 2 the responses were reduced by 75%. 4. Concentration-response relationships were studied for ATP and 2-MeSATP in the presence of L-NAME. It was found that L-NAME did not significantly inhibit the relaxant responses to these drugs. 5. Concentration-response relationships for ATP and noradrenaline were studied in the presence of reactive blue 2 (20 microM); the P2y-antagonist significantly inhibited the relaxant response to ATP, but not that to noradrenaline. 6. The distribution of nitric oxide synthase in rat pyloric sphincter was investigated immunohistochemically,with immunoreactive nerve fibres found throughout the circular muscle layer and myenteric plexus of the sphincter.7. While abundant vasoactive intestinal polypeptide (VIP)-containing nerve fibres were demonstrated immunohistochemically in the pyloric sphincter, relaxations to VIP (1 nM-0.3 micro M) were not observed in this preparation.8. It is concluded that ATP, acting through P2y-purinoceptors, and NO contribute to NANC inhibitory neurotransmission in rat pyloric sphincter. NO appeared to contribute to the later component of NANCrelaxation. The action of ATP was not mediated by NO, and VIP did not contribute to the NANCinhibitory responses in this preparation.