We and others have recently defined that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) functions as a negative regulator of platelet-collagen interactions involving the glycoprotein VI/Fc receptor gamma chain (GPVI/FcR-γ chain) signaling pathway.^, In this study, we hypothesized that PECAM-1 may be physically and functionally associated with FcγRIIa on the platelet membrane. The functional relationship between PECAM-1 and FcγRIIa was assessed by determining the effect of anti-PECAM-1 monoclonal antibody Fab fragments on FcγRIIa-mediated platelet aggregation and heparin-induced thrombocytopenia (HITS)-mediated platelet aggregation. Preincubation of washed platelets with monoclonal antibody fragments of 2BD4 directed against PECAM-1 and IV.3 directed against FcγRIIa completely blocked FcγRIIa-mediated platelet aggregation and HITS-mediated platelet aggregation, whereas anti-CD151 antibody had no blocking effect. Coengagement of FcγRIIa and PECAM-1 resulted in negative regulation of FcγRIIa-mediated phospholipase Cγ2 activation, calcium mobilization, and phosphoinositide 3-kinase-dependent signaling pathways. In addition, the physical proximity of FcγRIIa and PECAM-1 was confirmed by using fluorescence resonance energy transfer and coimmunoprecipitation studies. These results indicate that PECAM-1 and FcγRIIa are colocalized on the platelet membrane and PECAM-1 down-regulates FcγRIIa-mediated platelet responses. (Blood. 2003;102:3637-3645)