A simplified method for the assay of antithrombin III (AT) with the highly reactive thrombin substrate 2AcOH X H-D-CHG-Ala-Arg-pNA (substrate Th-1) is described. The assay may be performed at either 30 degrees C or 37 degrees C, and alternatively with the substrate H-D-Phe-Pip-Arg-pNA (S-2238). The standard curve is linear in the 12.5-150% range. For routine assays, 3 standard dilutions of plasma are sufficient, and these may be stored at -20 degrees C for 3 weeks. As only the test plasma must be diluted prior to the assay procedure, the test is more rapidly performed than previous manual assays. In 80 patients plasma samples, with AT in the 19-108% range, there was a high correlation with the results of immunoquantification (r = 0.96). There was also a high correlation between the results obtained with the manual method and the automated version described using the Cobas-Bio Centrifugal Analyser and substrate Th-1 (r = 0.96). Low AT levels in hereditary deficiency (particularly during heparin treatment), in liver cirrhosis, in disseminated intravascular coagulation (DIC), and heparin-treated thrombosis were confirmed.