A large body of evidence suggests that nitric oxide (NO) and ATP act as neurotransmitters in the regulatory mechanisms concerning several autonomic functions at the level of both the hypothalamus and the brain stem. In the present study, we investigated whether neuronal NO synthase containing neurones also express P2X(2) receptor subunit of the ATP-gated ion channel via double-labelling fluorescence immunohistochemistry. Our data demonstrate that a high percentage of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the rostral ventrolateral medulla (98%) and supraoptic nucleus of the hypothalamus (92%). Significant numbers of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the subpostremal (48%) and commissural (65%) subdivisions of the nucleus tractus solitarius. In the caudal ventrolateral medulla and raphe obscurus, 96% and 89%, respectively, of neuronal NO synthase containing neurones also express P2X(2) receptor subunit. In contrast to the supraoptic nucleus, there was a lower percentage of co-localisation between NO synthase and P2X(2) receptor subunit in the paraventricular nucleus of the hypothalamus. In summary, this study demonstrates for the first time that there is a widespread co-localisation of neuronal NO synthase and P2X(2) receptor subunit in the hypothalamus and brain stem of the rat. Further studies are required to elucidate whether NO and ATP functionally interact within the hypothalamus and the brain stem.