Purinergic P2X3 and P2X2/3 receptors mediate nociception. ATP is the most potent activator of P2X receptors. Muscle contraction increases interstitial ATP concentration. ATP excites group III and IV muscle nociceptors, and intramuscular administration in man induces strong muscle pain. The role of P2X receptors in neck muscle nociception was addressed in electrophysiological experiments in 119 C57BL/6 mice under general barbiturate anaesthesia. A,b-methylene ATP was bilaterally administered into semispinal neck muscles. Changes in brainstem nociception were measured by the jaw-opening reflex (JOR). The JOR was elicited by electrical stimulation of the tongue musculature and recorded in the digastric muscle. Within 2 hours after injection of 100 nmol/l or 1 mmol/l ATP, JOR integrals increased by 114% or 328%, respectively. Preceding intramuscular administration of the P2X receptor antagonists PPADS, A-317491, NF110, or NF449 prevented the ATP effect in a dose-dependent manner. Subsequent application of PPADS (100 nmol/l) reversed the ATP effect. Preceding or subsequent (100 nmol/l) intramuscular injection of TTX prevented or reversed the ATP effect. ATP induces sustained facilitation of craniofacial nociception by prolonged excitation of P2X receptors in neck muscles. Receptor profiles of P2X antagonists, inhibitory effects of subsequent administration, and suppression of ATP effect by blockade of myelinated afferents via TTX point to an important role of the heteromultimeric P2X2/3 receptor in neck muscle nociception.