A number of thrombin mutants have been constructed to investigate the role of Trp96 and the β-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr60APro60BPro60CTrp60D Asp60ELys60F of the β-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp96→Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ∼3 times more slowly than thrombin, with the two β-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ∼3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 μM−1 s−1 respectively, compared to 10 and 2.5 μM−1 s−1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased ∼6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the β-insertion loop is important for thrombin activity. But the mutation of Trp96→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro60BPro60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.