Summary. The platelet P2X1 purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X1 in platelet function is unknown, though stimulation with the P2X1 agonist α,β-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X1 stimulation on platelet activation. Stimulation of P2X1 with α,β-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to α,β-Me-ATP alone was less than that observed in heparin; however, α,β-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X1 antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by α,β-Me-ATP. Overall, this study demonstrates that P2X1 stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X1 stimulation, supporting a role for this receptor in regulating platelet activation.