Neck muscle nociception mediated by P2X receptors probably plays a key role in the pathophysiology of tension-type headache. Nociceptive input from semispinal neck muscles was investigated by bilateral injection of ATP (100 nmol/l, 20 ml) in anesthetized mice (n=59). The impact of neck muscle noxious input on brainstem nociceptive processing was monitored by the jaw-opening reflex (JOR) elicited by electrical tongue stimulation. Within two hours after ATP administration the JOR integral significantly increased by 112%. In order to investigate the role of P2X receptors in facilitation, the specific P2X receptor antagonists NF110 (P2X3, P2X2/3; 2–50 nmol/l; 20 ml), NF449 (P2X1, P2X2/3; 0.15–2 nmol/l; 20 ml), or A-317491 (P2X3, P2X2/3; 0.1–10 mM; 20 ml) were intramuscularly applied 15 min before ATP injection. All P2X antagonists suppressed the ATP effect in a dose-dependent manner. These results demonstrated that ATP induced long-term facilitation of brainstem nociceptive processing via heteromultimeric P2X2/3 receptors on neck muscle afferents. This purinergic mechanism may be involved in the pathophysiology of tension-type headache. Supported by grants of the German Headache Consortium (BMBF, 01EM0516).