We have examined the effects of purified human alpha-thrombin on factor VIII antigen (FVIII-Ag) release by human umbilical vein endothelial cells in culture. Alpha-thrombin induced a time and dose-dependent release of FVIII-Ag into supernatant medium. Alpha-thrombin-mediated FVIII-Ag release was not dependent on protein synthesis and was observed in both serum-free and serum-containing media. FVIII-Ag release, however, was prevented when the serine esterase activity of thrombin was inhibited. Pretreatment of human endothelial cells with alpha-thrombin, but not diisofluorophosphate-thrombin, prevented subsequent FVIII-Ag release by alpha-thrombin. Thrombin-mediated FVII- Ag release was not associated with significant 51Cr release from prelabeled endothelial monolayers. We conclude that alpha-thrombin induces release of preformed FVIII-Ag from human umbilical vein endothelial cells by a receptor-independent, nonlytic mechanism requiring serine esterase activity.