The inhibition of activated human Factor X by human plasma protease inhibitors was investigated in both purified and plasma systems. In the former, antithrombin III, alpha 1-proteinase inhibitor, and alpha 2-macroglobulin, at normal plasma concentrations, markedly inhibited activated Factor X. Significant inhibition by alpha 2-antiplasmin, however, was only achieved when present at 40 times its plasma concentration. The relative rates of inhibition of activated Factor X coagulant activity in normal human plasma, antithrombin III-deficient plasma, and alpha 1-proteinase inhibitor-deficient plasma were 1.0, 0.63, and 0.75, respectively. From these relative rates of inhibition and their measured concentrations in the 3 plasmas, it was calculated that antithrombin III, alpha 1-proteinase inhibitor, and alpha 2-macroglobulin contribute 53, 35, and 12%, respectively, to the inhibition of activated Factor X in normal human plasma. Using 125I-labeled activated Factor X and a combination of sodium dodecyl sulfate disc gel electrophoresis and immunoadsorption, it was then demonstrated that antithrombin III accounted for 45-55%, alpha 1-proteinase inhibitor, 35-40%, and alpha 2-macroglobulin, 10-15% of the inhibition of the labeled protease in normal human plasma. The values obtained with the deficient plasmas are consistent with the distribution of activated Factor X-inhibitor complexes in normal human plasma. These data show by two independent techniques, one measuring coagulant activity and the other 125I-labeled activated Factor X-inhibitor complexes, that both antithrombin III and alpha 1-proteinase inhibitor are the major inhibitors of activated Factor X in normal human plasma.