A three-step spectrophotometric assay was developed for measuring functional protein C (PC) in human plasma. The assay is based on: (1) adsorption of citrated platelet-poor plasma on barium citrate and elution of the vitamin K-dependent factors with EDTA; (2) activation of PC by incubation of the mixture of vitamin K-dependent factors with a complex of thrombin and its endothelial cell cofactor, thrombomodulin; (3) addition of antithrombin III and heparin to the system to inhibit thrombin and other coagulation enzymes generated during incubation and measurement of the activated PC with a synthetic (chromogenic) substrate. The assay appears to be specific for PC because: (a) PC- depleted plasma (by immunoadsorption) is inactive; (b) addition of purified PC to PC-depleted plasma reconstitutes its activity; and (c) no enzymatic activity is generated in the absence of the thrombin- thrombomodulin complex. Mixtures of a normal plasma pool with PC- depleted plasma yielded an amount of enzymatic activity proportional to the fraction of normal plasma. Using this as a standard curve, the amount of PC in the plasma of 23 normal subjects was 97% +/- 15%. The within-assay coefficient of variation was 3.5% and the between-assay coefficient 6.5%. A linear correlation (r = 0.86) was found between PC as measured with the functional assay and with a radioimmunoassay. In 3 patients with congenital PC deficiency, the functional PC level was 37% +/- 9% and the antigen level 64% +/- 11%. It is concluded that the present assay may be used for reliable and accurate estimation of activatable PC in human plasma.