Synthetic substrates have recently been developed for the assay of several of the proteolytic enzymes required for blood coagulation, One of these substrates, S-2238, is specific for thrombin and hence can also be used to measure antithrombin-heparin co-factor (AT-III). The thrombin remaining after neutralization by AT-III cleaves a chromophore, p-nitroaniline, from the substrate, which can then te quantified in a spectrophotometer. This new assay for AT-III inhibitory activity was evaluated for normal subjects and for patients who had certain pathologic conditions associated with reduced levels of AT-III. Results from this assay were compared with those from AT-III assays by Laurell immunoelectrophoresis (an immunologic method) and antithrombin inhibition of factor Xa (a biologic clotting method). The chromogenic assay using S-2238 was found to be sensitive, accurate, and easy to perform; its results for both normal subjects and patients correlated extremely well with those of the immunologic and factor Xa inhibition assays (r = 0.94-0.98). Measurement of AT-III using this synthetic substrate should be a valuable addition to routine assays in a clinical coagulation laboratory. These and future substrates may prove extremely useful for assays of factors involved in blood coagulation and fibrinolysis.