An orphan G protein-coupled receptor, termed 6H1, with approximately 30% sequence identity to P2Y receptors has been proposed to be a P2Y receptor (p2y5) based solely on a radioligand binding assay with [35S]dATP alphaS [Webb et al. (1997) Biochem. Biophys. Res. Commun. 219:105-110]. Previous work in our laboratory has shown that [35S]-dATP alphaS is not a general ligand for P2Y receptors, and thus inclusion of the p2y5 receptor in the family of P2Y receptors is questionable. To define unambiguously whether the p2y5 receptor is a P2Y receptor, we have cloned the turkey homologue of the chick p2y5 receptor. Sequence analysis indicated that the turkey receptor contains an additional 32 amino acids at its carboxy terminus compared to the published chick sequence. HA epitope-tagged turkey p2y5 receptors were stably expressed in 1321N1 human astrocytoma cells, and cells shown to express the HA-tagged p2y5 receptor by an intact cell-based ELISA were used to determine whether changes in second messenger levels occurred in response to a series of nucleotides. ATP, ADP, UTP, UDP, dATP alphaS, and A2P4 had no effect on either inositol phosphate or cyclic AMP concentrations in cells expressing the p2y5 receptor. Robust inositol phosphate and cyclic AMP responses occurred to other G protein-coupled receptors expressed in 1321N1 cells, which indicate that these cells contain all of the necessary signaling components to generate these second messenger responses. These data indicate that the 6H1/p2y5 receptor is not a member of the P2Y receptor family of signaling proteins.