Antithrombin (AT) is a single chain plasma glycoprotein that belongs to a superfamily of serine protease inhibitors (Serpin). AT is the major inhibitor of the serine proteases of the coagulation cascade, notably thrombin and factor Xa. In addition, AT has also been demonstrated to have anti-inflammatory properties. Recently, cleaved and latent forms of AT have been shown to function as antiangiogenic agents. However, the molecular mechanisms by which native AT and cleaved AT exert their anti-inflammatory and antiangiogenic effects remains unknown. In this study, we have investigated the effects of both native and cleaved forms of AT on endothelial cell proliferation and nuclear factor κB (NF-κB) activation in the endothelial cells. Bovine pulmonary artery endothelial cells (BPAE) was used in the study. Human α-antithrombin was purified from plasma by affinity chromatography on heparin Sepharose, followed by anion-exchange chromatography. Cleaved form of AT was prepared by digestion of purified human AT with human neutrophil elastase, followed by chromatography on a heparin Sepharose column. The endothelial cells were stimulated with basic fibroblast growth factor (bFGF) or tumor necrosis factor-α (TNF-α) in culture and incubated with either native AT or cleaved AT. Endothelial cell proliferation was measured by the MTT (methylthiazolyldiphenyl-tetrazolium bromide) cell proliferation assay as well as by counting cell numbers before and after the treatment. The results demonstrate that both native AT and cleaved AT could inhibit bovine pulmonary artery endothelial cell proliferation. These results contrast with those reported with other types of endothelial cells such as human umbilical vein endothelial cells (HUVEC) in which only cleaved form but not native form of AT inhibits cell proliferation. NF-κB activation was detected by the ELISA-based assay using antibodies specific for the activated form of p50 and p65 subunit of the NF-κB. In native AT, the inhibitory activity of endothelial cell proliferation was associated with down-regulation of NF-κB as measured by decreased nuclear p65. However, the cleaved AT showed minimal effect on the NF-κB activation. Our results suggest that although both native and cleaved AT inhibit endothelial cell proliferation, they might use different signal transduction pathways. Moreover, our findings suggest that native and cleaved AT may have differential effects on different types of endothelial cells.