OBJECTIVE: A single kindred in North America with venous thrombosis was described as having defective fibrinolysis because of increased levels of plasminogen activator inhibitor-1 (PAI-1). Our study describes the discovery of protein S deficiency in this kindred and its association with venous thromboembolism. DESIGN: A family study. SETTING: Community. PARTICIPANTS: Twenty-eight adults (ages 21 to 71 years) from three generations of the kindred; seven had a history of venous thromboembolism. MEASUREMENTS: Plasma levels of total and free protein S antigen, as well as the activities of protein S, protein C, PAI-1, and antithrombin III. RESULTS: Six of 7 persons (86%) with a history of venous thromboembolism were deficient in total and free protein S; of 21 asymptomatic members, 9 were deficient in protein S (P = 0.08). When compared with these 9 asymptomatic family members, the 6 persons with protein S deficiency and a history of thrombosis tended to smoke (P = 0.01) and to have higher triglyceride levels (P = 0.001). Overall, the mean PAI-1 activity in the 7 persons who had thrombosis was 7.9 kAU/L (AU/mL) and was 9.3 kAU/L (AU/mL) in the 21 persons who did not have thrombosis (95% CI, -9.9 to 7.0). CONCLUSIONS: In this kindred, a deficiency of total and free or functional protein S is the cause of thrombosis. Measurement of PAI-1 activity was not useful in the evaluation of familial thrombosis. The utility of the routine measurement of PAI-1 activity in the evaluation of familial thrombosis has not been established.