Cimex lectularius salivary gland homogenate delayed the recalcification time of human citrated plasma. Separation of the salivary gland homogenate by molecular sieving HPLC chromatography resulted in a single major peak of anticlotting activity with an apparent molecular mass of 17,000. The anticoagulant principle inhibited the activation of factor X to factor Xa in the tenase complex (FVIII, FIXa, FX, phospholipids, and calcium). However, it did not directly inhibit already activated factor Xa, suggesting that the anticlotting activity is not an anti-factor Xa. Additionally, this salivary gland anticoagulant further retarded the recalcification time of factor VIII- and factor IX-deficient plasmas, suggesting that the anticlotting principle is not directly inhibiting either the coagulation factor VIII or factor IXa. Altogether these data suggest that the anticlotting activity is an inhibitor of the activation of factor X to factor Xa in the tenase complex.