The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocyto-chemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X 1 and P2X 5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X 1 and P2X 5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X 1 and the kinetics of P2X 5. When expressed in Xenopus oocytes, hetero-oligomeric P2X 1؉5 ATP receptors were characterized by slowly de-sensitizing currents highly sensitive to the agonist ␣,␤-methylene ATP (EC 50 ‫؍‬ 1.1 M) and to the antagonist trinitrophenyl ATP (IC 50 ‫؍‬ 64 nM), observed with neither P2X 1 nor P2X 5 alone. Direct physical evidence for P2X 1؉5 co-assembly was provided by reciprocal subunit-specific co-purifications between epitope-tagged P2X 1 and P2X 5 subunits transfected in HEK-293A cells.