Antithrombin III (ATIII) has been expressed in transiently transfected COS-1 monkey cells and in stably transformed Chinese hamster ovary cells, and the resultant protein has been characterized for biological activity. Both cell types efficiently secrete high levels of heterogeneous molecular weight forms of ATIII antigen. The heterogeneity results from differences in post-translational modifications. However, only a small percentage (5-10%) of the total antigen expressed is biologically active. The fraction of biologically active ATIII has been purified from total ATIII by affinity fractionation on heparin-Sepharose. This fractionation indicates that the differences in the active and inactive forms of expressed ATIII result from differences in their ability to bind heparin. Purified ATIII has a specific activity very similar to that of plasma-derived ATIII and exhibits typical heparin-accelerated ATIII activity. The biologically active fraction of ATIII appears to represent the higher molecular weight forms of the ATIII expressed and is likely not a result of altered asparagine-linked glycosylation; however, the nature of the post-translational modification required for ATIII activity remains unclear. The ability to express biologically active ATIII at such high levels should allow further investigations of the structural requirements for ATIII activity.