The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.