Human antithrombin III cDNA was cloned into an expression vector suitable for transient expression in COS cells. Upon transfection COS cells secreted a single immunoreactive 58-kDa protein. Quantitation of secretion levels by ELISA indicated that at 44 hr posttransfection cells were secreting 48 +/- 5 ng of antithrombin III per 10(6) cells per 24 hr. Heparin-agarose chromatography resulted in the elution of the COS-derived protein as a broad band between 0.3 and 1.0 M NaCl. 35S-labeled medium from transfected cells reacted with human thrombin (1.5 ng/ml) in the absence of heparin. In 40 min, greater than 80% of the immunoreactive material was found as a higher molecular weight species, consistent with stoichiometric covalent complex formation. In a two-stage chromogenic thrombin inactivation assay, under pseudo-first-order conditions, at 16 nM antithrombin III the t1/2 was 74 min and 50 min for plasma and COS cell-derived antithrombin III, respectively, in the absence of heparin. In the presence of 17.4 nM high-affinity heparin, the t1/2 was 5.2 min and 2.2 min, respectively.