The P2Y₁ ADP receptor activates Gq and causes increases in intracellular calcium through stimulation of phospholipase C. In this study, we investigate the role of the amino acid residues in the carboxyl terminus of the human P2Y₁ receptor in Gq activation. Stimulation of CHO-K1 cells stably expressing the wild type human P2Y₁ receptor (P2Y1-WT cells), P2Y₁-R340-L373, or P2Y1-D356-L373 with 2-MeSADP caused inositol phosphate production. In contrast, P2Y₁-T330-L373, cells expressing a mutant lacking the entire C-terminus, completely lost its response to 2-MeSADP. Similar data were obtained in these cell lines when calcium mobilization was measured upon stimulation with 2-MeSADP, indicating that the 10 amino acids (³³⁰TFRRRLSRAT³³⁹) in the C-terminus of the human P2Y₁ receptor are essential for Gq coupling. Radioligand binding demonstrated that both the P2Y₁-WT- and P2Y₁-T330-L373- expressing cells have almost equal binding of [³H]MRS2279, a P2Y₁ receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y₁₂ receptor with the P2Y₁ carboxy terminus failed to elicit Gq functional responses, indicating that the P2Y₁ carboxy terminus is essential but not sufficient for Gq activation. Finally, the cells expressing a double mutant P2Y₁ receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R³³³R³³⁴) in the C-terminus of the human P2Y₁ receptor are essential for Gq coupling.