Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow → fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel β-strands of the B-chain (Met180-Tyr184a, Lys224-Tyr228, and Val213-Gly219) diagonally crossed by the Glu188-Glu192 strand. The site is shaped further by the loop connecting the last two β-strands and is located more than 15 Å away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp189 of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr184a, Arg221a, and Lys224, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg221a (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.