The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.