Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.