A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5'-triphosphate (UTP) formed a nucleobase pi-pi complex with conserved Phe3.32. Selectivity-enhancing 2'-amino-2'-deoxy substitution interacted through pi-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A "sequential ligand composition" approach for docking the flexible dinucleotide agonist Up4U demonstrated a shift of conserved cationic Arg3.29 from the UTP gamma position to the delta position of Up4U and Up4 ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2'-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC50 = 8 nM) was 300-fold P2Y2-selective versus P2Y4. 2'-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y2 potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y2 receptor recognition suggests mutations for model validation.