Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.