Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that plays a pivotal role in the regulation of hemostasis. The cDNA for ATIII has been available, but genetic studies on the functional domains of ATIII have not progressed because of the absence of an expression system that will yield sufficient quantities of biologically active protein for biochemical analyses. In the present studies the cDNA of the human antithrombin III gene was inserted into the vector pVL 1393, which is suitable for cotransfection of Spodoptera frugiperda (Sf9) insect cells with Baculovirus wild-type DNA. Recombinant virus particles were selected by the presence of occlusion-negative plaques. Upon infection with purified recombinant virus, Sf9 cells secreted 10-35 micrograms of ATIII/1 x 10(6) cells. Southern analysis of DNA from infected cells demonstrated incorporation of the full-length cDNA into the Baculovirus recombinant, and RNase protection experiments verified the presence of full-length transcript. This recombinant ATIII protein was immunologically reactive with antisera raised against native human ATIII, formed stable complexes with thrombin, and was heparin-accelerated at the same concentration as native human ATIII. In addition, the recombinant ATIII retained specificity for the same molecular species of heparin that activates authentic human ATIII. This is the first successful production of active, recombinant ATIII in quantities that will allow purification on the milligram scale and permit a biochemical analysis of genetically engineered variants.