The effect of A-317491 (5-([(3-Phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid), a recently described selective P2X3 and P2X(2/3) receptor antagonist, on inflammatory mechanical hyperalgesia was examined. In the rat Freund's complete adjuvant model of inflammatory pain, s.c. administration of A-317491 dose-dependently reversed mechanical hyperalgesia. Maximum percent reversal (72%) was seen 3 h after administration at 10 mg/kg. Substantial plasma concentrations were measured for A-317491 after s.c. dosing 3, 10 and 30 mg/kg. However, the brain-to-plasma concentration ratio, determined 1 h after a 10 mg/kg s.c. dose, indicated limited penetration of A-317491 into the central nervous system. As revealed by neural activity recorded from single C-fiber nociceptive afferent in a Freund's complete adjuvant-inflamed rat skin-nerve preparation, topical application of A-317491 completely blocked afferent activation and mechanical sensitization induced by alpha,beta-methylene ATP, a P2X agonist. These results suggest that A-317491 is a peripherally acting P2X blocker. Its efficacy demonstrates the importance of peripheral P2X3/P2X(2/3) receptors in mediating ATP-associated mechanical hyperalgesia following inflammation, confirming previous suggestions of a significant role for P2X(2/3).