Vitamin K-deficient animals and humans developed a more severe coagulopathy when treated with vitamin E, which was due to further reduction in the vitamin K-dependent coagulation factors (II, VII, IX, and X). This phenomenon was not seen in normal vitamin K-sufficient animals or human subjects. The mechanism by which vitamin E causes this effect is not known. These coagulation factors are produced by the liver in precursor forms and are converted to functional proteins by a vitamin K-dependent reaction. Analysis of one of these coagulation factors, prothrombin (factor II), in plasma of vitamin K-deficient animals and humans treated with vitamin E was done in this study. The precursor of factor II is antigenically similar to biologically active factor II and can be activated to form thrombin by Echis carinatus venom. The data showed that functional factor II coagulant activity was reduced below base in warfarin-treated humans and animals given vitamin E. Factor II antigen as determined by electroimmunoassay in humans and factor II coagulant activity as measured using Echis venom in animals were unchanged and no different from untreated controls. The data suggest that vitamin E acts at the vitamin K-carboxylase step of carboxylation of precursor prothrombin and not in the synthesis of the precursor protein.