Traces of activated factor VII (FVIIa) are required to maintain haemostasis. Activated factor X (FXa) is the main activator of FVII in the absence of tissue factor. However, little is known about how this mechanism is regulated. We and others reported the interaction between FVII and the endothelial cell protein C receptor (EPCR). We have analysed the role of EPCR in the FXa-dependent FVIIa generation. Activation was performed on the surface of human aortic endothelial cells in the presence or absence of a blocking anti-EPCR monoclonal antibody (mAb). Western-blot analyses revealed that FVII activation was increased twofold upon EPCR blocking. Kinetic analyses revealed that blocking doubled the catalytic efficiency for activation. Protein C was unable to mimic the effect of the anti-EPCR mAb on activation. Surface plasmon resonance experiments revealed that binding of EPCR and phospholipids to FVII were mutually exclusive. The 50% inhibitory concentration value for phospholipids to reduce the binding of FVIIa to EPCR was 57.67 +/- 0.11 micromol/l. Immunofluorescence experiments showed that EPCR and phosphatidylserine are located at different regions of the cell surface. We propose that EPCR downregulates FVII activation by moving it from phosphatidylserine-rich regions. In summary, this study described a new anticoagulant role for EPCR.