A potent and specific inhibitor of the human coagulation protease thrombin was identified in salivary gland extracts of the tsetse fly, Glossina morsitans morsitans, an important vector of African trypanosomiasis. This low molecular weight peptide (MW = 3,530 Da as determined by laser desorption mass spectrometry) was purified using a combination of size-exclusion chromatography and reverse-phase, high-performance liquid chromatography, respectively. Amino terminal sequencing of the purified protein reveals no homology to any previously identified serine protease inhibitor or naturally occurring anticoagulant. The tsetse thrombin inhibitor (TTI) is a stoichiometric inhibitor of thrombin, with an apparent equilibrium dissociation inhibitory constant (Ki*) [corrected] of 584 x 10(-15)M. In addition, it is also a potent inhibitor of thrombin-induced platelet aggregation. Like other hematophagous arthropods, tsetse flies appear to have evolved a novel protease inhibitor capable of antagonizing host hemostasis and facilitating blood feeding.