Salivary gland extracts from the yellow fever mosquito Aedes aegypti contain a factor Xa-specific anticoagulant detectable in vitro by clotting assays. The anticoagulant is found only in female salivary glands, which is consistent with its role in bloodfeeding. Extracts of adult female glands significantly delay both the prothrombin time and the activated partial thromboplastin time, but have no effect on the thrombin clotting time. The anticoagulant has been shown to result from the specific inhibition of coagulation factor Xa based on a FXa clotting assay and the inhibition of FXa-directed cleavage of the synthetic chromogenic substrate, chromozym X. The inhibition of FXa by female salivary gland extracts exhibits noncovalent, noncompetitive inhibition kinetics and is reversible. The anticoagulant is destroyed by boiling for 10 min or heating at 56 degrees C for 30 min, has a 4 mM calcium optimum with no magnesium requirement, and has a pH optimum of 8.0. The anticoagulant activity has an apparent molecular weight of 35.5 kDa, as determined by molecular sieve. We propose that the A. aegypti anticoagulant is a novel, proteinaceous serine protease inhibitor specific for FXa.