P2Y receptors are G protein-coupled receptors stimulated by extracellular nucleotides. Both the P2Y 1 and the P2Y 6 receptors are preferentially activated by nucleoside 5′-diphosphates, but favor different base moieties. In the case of the P2Y 1 receptor the preferred base is adenine, while the P2Y 6 receptor is activated by uracil nucleotides. To identify potential amino acid domains that interact with the base moiety, we used a chimeric receptor approach, employing the human P2Y 1 receptor as core structure to investigate the role in receptor activation of extracellular loops (ELs) and transmembrane domains (TMs) of the rat P2Y 6 receptor. The chimeric receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C (PLC) induced by the potent P2Y 1 receptor agonist 2-MeSADP or the potent P2Y 6 receptor agonist UDP. Replacement of the N-terminus or EL2 resulted in low (∼50μM) potency of the agonist 2-MeSADP, thus confirming the importance of EL2 in ligand recognition. Upon replacement of several regions, the potency of the P2Y 1 agonist 2-MeSADP was either 1–2μM (N-terminus and EL1, or EL1 and EL3) or 72μM (N-terminus and EL3). Concurrent replacement of three regions (N-terminus, EL1, and EL3) completely precluded activation by 2-MeSADP. Our study identified domains of the P2Y 6 receptor that contribute to receptor activation by UDP and hence seem to be involved in uracil recognition. Upon replacement with extracellular domains of the P2Y 6 receptor sequence we observed a trend toward gain of receptor-induced PLC activation by UDP, for example, in the chimera containing replacements of both the N-terminus and EL1. Exchange of three receptor domains led to a construct with an EC 50 value for UDP of 19μM and a maximal inositol phosphate accumulation similar to the native P2Y 6 receptor. Within receptor constructs of combined domain exchanges the additional substitution of Tyr 110 by the corresponding Asn from the P2Y 6 receptor showed a significant increase for activation by UDP, but only when combined with the N-terminal domain and TM1. The residue Tyr 110 was identified to play an important role in the recognition of the nucleobase in the P2Y 1 and P2Y 6 receptors.