Protein S is an antithrombotic plasma protein that serves as a cofactor for another plasma protein, activated protein C. Protein S is required for the expression of the anticoagulant effect of activated protein C, which inhibits blood clotting at the levels of factors V and VIII in the blood-clotting cascade. We postulated that patients deficient in protein S would have inadequate regulatory control of the clotting cascade and would be prone to thrombotic disease in a manner similar to that of patients congenitally deficient in protein C. To determine whether protein S deficiency is associated with recurrent thrombosis, we developed a functional assay for the plasma protein. With this assay, the protein S activity of normal persons ranges from 63 to 160 per cent. Using this test, we have now identified six unrelated persons with severe recurrent venous thrombosis who were deficient in protein S, with levels between 15 and 37 per cent while they were not receiving warfarin therapy. Our data suggest that the determination of protein S levels will be useful in the evaluation of patients with recurrent thrombosis.