The predominant mode of the anticoagulant action of heparin is considered to be the enhancement of the rate of inactivation, by antithrombin-III, of several activated clotting factors. Recent evidence, however, suggests that heparin can reversibly inhibit the activation of prothrombin and factor X even in the absence of anti- thrombin-III. This antithrombin-III-independent action of heparin has been demonstrated only in purified clotting factor systems. In order to determine the significance of the antithrombin-III-independent effects of heparin in plasma, the effects of heparin on the activation of factor X and prothrombin were studied in antithrombin-III- depleted plasma produced by affinity chromatography of normal plasma on heparin-Sepharose. Heparin partially inhibited the activation of factor X and prothrombin on the addition of either factor IXa or factor Xa in anti- thrombin-III-depleted plasma. This inhibition was demonstrable only when high concentrations (1 or 10 units/ ml) of heparin were used. In contrast, when as little as 1% antithrombin-III was added to antithrombin-III-depleted plasma containing 1.0 u of heparin per ml of plasma, no factor X or prothrombin activation could be demonstrated. Thus, it appears that in comparison with the magnitude of the antithrombin-III-dependent effect, the contribution of the antithrombin-III-independent anticoagulant effect of heparin on the activation of factor X and prothrombin in normal plasma is limited.