Administration of a,b-meATP (ATP) into neck muscles facilitates brainstem nociception in anesthetized mice. The unspecific nitric oxide synthase (NOS) blocker L-NMMA inhibits and reverses this facilitation. The present study addressed hypothesized involvement of the neuronal NOS (nNOS) isoenzyme in ATP-mediated neck muscle facilitation. Bilateral infusion of ATP (1 mM, 25 ml; Sigma- Aldrich, USA) into semispinal neck muscles was performed in anesthetized mice (n=18). Impact of neck muscle nociception on sensory brainstem processing was electrophysiologically monitored via the jaw-opening reflex (JOR). The JOR was elicited by electrical tongue stimulation and monitored for at least 90 min after ATP infusion (control). Different dosages of highly selective nNOS inhibitor NPLA (0.5, 1, 2 mg/kg, Tocris, USA) were intraperitoneally injected 30 min before or 90 min after ATP infusion. After sole ATP infusion JOR increased by 20318 % (meansem, p<0.001). Preceding NPLA (2 mg/kg) prevented facilitatory ATP effect for at least 90 min. After preceding injection of 0.5 mg/kg and 1 mg/kg NPLA ATP effect decreased to 9512 % and 567 %, respectively. Subsequent application of 2 mg/kg NPLA did not reverse established ATP-evoked reflex facilitation. ATP reliably induced long-term facilitation of neck muscle nociception. Preceding NPLA application blocked this facilitation in a dose- dependent manner. Subsequent nNOS inhibition did not affect reflex facilitation. Thus, nNOS probably plays a role in induction but not maintenance of nociceptive facilitation in the brainstem. Inducible or endothelial NOS isoenzymes are putative candidates for effect maintenance. These results point to a major role of NOS isoenzymes in neck muscle nociception.