The P2Y family of G-protein coupled receptors are activated by adenine and uridine di- and triphosphate nucleotides and nucleotide sugars and are implicated in a wide range of important human physiologies. Difficulty studying these receptors and in their successful manipulation as therapeutic targets has historically derived from a lack of available pharmacological tools that discriminate among members of the P2Y receptor family. The studies described here focus on the P2Y1 receptor, a key mediator of ADP-induced platelet aggregation. Based on the structure of the recently synthesized, high-affinity P2Y1 receptorselective antagonist, 2-iodo-N6-methyl-(N)-methanocarba-2 -deoxyadenosine-3´,5´- bisphosphate (MRS2500), we undertook the development of a high-specific radioactivity radioligand for the P2Y1 receptor, suitable for the study of endogenous receptors in mammalian tissues and cell lines. Using an enzymatic phosphorylation reaction, we successfully generated [32P]MRS2500 with a specific activity of 9120 Ci/mmol. The selectivity and affinity of [32P]MRS2500 for the P2Y1 receptor were confirmed in radioligand binding assays with Sf9 insect cell membranes overexpressing recombinant P2Y1 receptors. The utility of [32P]MRS2500 for the study of endogenous P2Y1 receptors was examined using washed human platelets and membranes prepared from various tissues of the adult rat. We applied this high-specific radioactivity radioligand to observe surface expression of P2Y1 receptor binding sites in human platelets and MDCK(II) epithelial cells following incubation with P2Y1 receptor agonists. In human platelets, the rapid, agonist-promoted desensitization of the P2Y1 receptor observed after incubation with the selective agonist (N)- methanocarba-2-methylthioadenosine-diphosphate (MRS2365) also occurs for the Gqcoupled 5-HT2A receptor after incubation of platelets with 5-hydroxytryptamine (5-HT). The rapid, agonist-promoted desensitization of the P2Y1 receptor of platelets was accompanied by a modest decrease (< 20%) in the number of surface [32P]MRS2500 binding sites and only a partial recovery of P2Y1 receptor responsiveness after removal of the selective agonist MRS2365. Platelets, therefore, appear to employ a unique mechanism for prolonged termination of P2Y1 receptor signaling in which desensitized receptors are maintained at the cell surface, unable to respond to subsequent agonist stimulation. In intact MDCK(II) cells overexpressing recombinant P2Y1 receptors, incubation with 2MeSADP was followed by a 50% loss of surface [32P]MRS2500 binding sites that was agonist-concentration dependent and required the formation of clathrin-coated pits. Mutagenesis studies indicated that this rapid, agonist-promoted loss of surface binding sites requires two serine residues, Ser352 and Ser354, in the receptor carboxyl terminus. The findings presented here indicate that P2Y1 receptor cell surface expression is regulated in an agonist-dependent manner that differs in at least two cell types and suggests an important role for phosphorylation in agonist-dependent desensitization and internalization of the P2Y1 receptor.