Regions of hirudin important for its inhibitory activity with thrombin have been examined by site-directed mutagenesis. Since thrombin has a primary specificity for basic amino acids, each of the three basic residues and the histidine in hirudin were mutated to glutamine. Mutation of Lys-47 caused a small increase (9-fold) in the dissociation constant whereas the other mutations were without effect. These results indicate that hirudin is different from most other inhibitors of serine proteases in that interactions with the primary specificity pocket of its target enzyme are not crucial to its inhibitory activity. The acidic nature of the carboxyl region of hirudin was found to be important for its interaction with thrombin. Single and multiple mutations of carboxyl-terminal glutamate residues (57, 58, 61, and 62) to glutamine caused increases in the dissociation constant. This value increased with the number of mutations and reached a maximum of 61-fold when all four glutamate residues were mutated. Kinetic studies indicated that in all cases where an increase in dissociation constant was observed, it was predominantly due to a decrease in the association rate constant.