High concentrations of adenosine-5′-diphosphate ADP are able to induce partial aggregation without shape change of P2Y₁ receptor-deficient mouse platelets through activation of the P2Y₁₂ receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse α_IIbβ₃ integrin, revealed a low level activation of α_IIbβ₃ in P2Y₁ receptor-deficient platelets in response to 100 μM ADP or 1 μM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of ³²P-labeled platelets showed that P2Y₁₂-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P₂₀) or pleckstrin (P₄₇) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y₁₂ receptor is able to trigger a P2Y₁ receptor-independent inside-out signal leading to α_IIbβ₃ integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the α_IIbβ₃ integrin, and (iii) the transduction pathway triggered by the P2Y₁₂ receptor is independent of PKC but dependent on phosphoinositide 3-kinase.