A radioimmunoassay (RIA) has been developed for the determination of antithrombin III (AT III) in man. The detection limit was 25 ..mu..g/dl. At III-RIA level and biological activity (anti-Xa) was significantly correlated (r = 0.737, P < 0.0001). Plasma levels in 36 healthy males (mean +- SD, 19.9 +- 2.5 mg/dl) and 21 healthy females (19.1 +- 2.4 mg/dl) were similiar. Serial AT III measurements in normal menstruating females showed lower levels during midcycle and higher concentrations during menstruation. In carcinomas, the AT III levels were lower than normal, particularly in hepatocellular carcinoma. In cirrhosis of liver, the levels were markedly decreased and in some patients were below that found in congenital AT III deficiency. Patients with deep vein thrombosis and patients with heart valve replacement had lower levels than normal, while patients with cerebral vascular occlusion had normal levels. The possible use of AT III as a diagnostic tool of post-operative deep vein thrombosis was demonstrated in one patient after hysterectomy. The increased sensitivity, specificity and precision of this type of assay offer distinct advantages over existing methods of AT III estimation.