Two subtypes of G protein-coupled receptors for nucleotides (P2U and P2Y purinoreceptors) contain several conserved positively charged amino acids in the third, sixth, and seventh putative transmembrane helices (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P2U and P2Y purinoceptors (P2UR and P2YR), we postulated that some of these positively charged amino acids are involved in binding of the negatively charged phosphate groups of nucleotides. To investigate the role of the conserved positively charged amino acids in purinoceptor function, a series of mutant P2UR cDNAs were constructed so that lysine 107 and arginine 110 in TMH 3, histidine 262 and arginine 265 in TMH 6, and arginine 292 in TMH 7 were changed to the neutral amino acid leucine or isoleucine. The mutated P2UR cDNAs were stably expressed in 1321N1 astrocytoma cells and receptor activity was monitored by quantitating changes in the concentration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P2UR agonists. Neutralization of His262, Arg265, or Arg292 caused a 100-850-fold decrease in the potency of ATP and UTP relative to the unmutated P2UR and rendered ADP and UDP ineffective. In contrast, neutralization of Lys107 or Arg110 did not alter the agonist potency or specificity of the P2UR. Neutralization of Lys289 in the P2UR, which is expressed as a glutamine residue in the P2Y subtype, did not alter receptor activity; however, a conservative change from lysine to arginine at this position altered the rank order of agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP. A three-dimensional model of the P2UR indicates the feasibility of His262, Arg265, and Arg292 interactions with the phosphate groups of nucleotides.