Activation of human prothrombin (Pro) occurs following two cleavages by membrane-bound factor Xa (fXa) (Arg271 followed by Arg320, generating prethrombin 2 as intermediate). Binding of factor Va (fVa) to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase (IIase). It has been established that IIase is a 1:1 stochiometric complex between fVa and fXa on a lipid surface. This complex catalyzes the activation of Pro following the opposite pathway (cleavage at Arg320 followed by Arg271, generating meizothrombin as intermediate). Thus, the activity of factor Xa within IIase is controlled by fVa. Most of the data published in the literature on IIase function was obtained using limiting amounts of fXa in the presence of saturating concentrations of fVa. It is always assumed that IIase acts as one enzyme and that fXa needs to be saturated with fVa. However, if IIase is one enzyme, theoretically reversing the concentrations of reactants will have no effect on the pathway and catalytic efficiency of the enzyme for cleavage of Pro. We have assessed the pathway to thrombin formation in the presence of high or low concentrations of fVa (with respect to fXa). In the absence of fVa, fXa (10nM) activates Pro slowly through initial cleavage at Arg271. Very little thrombin is formed under these conditions. In the presence of 10nM fXa and 100pM fVa there is a significant increase in the concentration of thrombin as demonstrated by the increase in the appearance of the B chain of thrombin. Under these experimental conditions Pro is activated through the pathway characterized by the appearance of prethrombin 2 as intermediate. No fragment 1·2-A was apparent. In the presence of 100pM fXa with 10nM fVa, Pro is activated exclusively through the pathway characterized by the appearance of meizothrombin as demonstrated by the presence of fragment 1·2-A and the absence of prethrombin 2. Kinetics assessments of thrombin generation using both conditions revealed that the kcat for thrombin generation by IIase assembled in the presence of low concentrations of fVa is ∼240 min−1 while the the kcat of IIase assembled with saturating concentrations of fVa is ∼2100 min−1. These data were verified by studying the activation of recombinant Pro that can only be cleaved at Arg320 (rMZ). In the presence of limiting concentrations of fVa, rMZ was cleaved with a rate that is approximately 7-10-fold slower than the rate of rMZ cleavage in the presence of saturating concentrations of fVa. Overall our data support the following hypothesis: in the presence of low concentrations of fVa that are generated during initiation of clotting, IIase activates Pro through initial cleavage at Arg271 (E271). As clotting occurs and more fVa is generated, a new form of IIase is formed following the binding of fXa to fVa and the interaction of the complex with Pro (E320). Under these conditions, while both forms of IIase exist, E320 that has higher catalytic efficiency activates Pro via initial cleavage at Arg320. Our data are in agreement with recent findings that have suggested that IIase activation of Pro may be best described as an ordered ping-pong reaction and imply that the concentration of fVa generated during clotting, dictates which form of IIase will first cleave Pro. In conclusion, our findings suggest that fVa shuttling between E271 and E320 directs Pro activation by factor Xa locally at the place of vascular injury.