Macromolecular substrate recognition and serine proteinase specificity lie at the heart of the tightly regulated hemostatic response. Mechanisms established for the less specific serine proteinases of digestion have played a dominant role in guiding investigations of the basis for the narrow specificities exhibited by the coagulation enzymes. These concepts have also dominated the development of specific inhibitors of coagulation for therapeutic purposes. Studies of the enzymology and physical biochemistry of prothrombinase challenge these prevailing ideas by establishing a principal role for exosites within the enzyme in determining substrate recognition and directing the action of the enzyme on its biological substrate. Mechanisms by which narrow protein substrate specificity is achieved by prothrombinase also apply to several other reactions of coagulation. These strategies are increasingly evident in the action of other families of enzymes that act with high specificity on protein substrates. Exosite-driven enzymic function probably represents a widely employed biological strategy for the achievement of high macromolecular substrate specificity.