The study investigates the synthesis of antihemophilic factor (AHF, Factor VIII) antigen by cultured human endothelial cells. Immunofluorescence studies using monospecific rabbit antibody to human AHF confirmed the presence of AHF antigen in endothelial cells, while control studies with smooth muscle cells and fibroblasts were negative. Radioimmunoassay demonstrated that cultured endothelial cells contain AHF antigen, which is released into the culture medium. Endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions from the culture media. The incorporation of radioactivity into immunoprecipitable protein was specific for AHF antigen, as it was not detected in smooth muscle cell media. However, despite the active synthesis of AHF antigen, no procoagulant activity was detected in the culture medium. Experiments using cycloheximide inhibited protein synthesis and [³H]leucine incorporation, but AHF antigen synthesis resumed after the inhibitor was removed. The study also found that exogenous AHF procoagulant activity was not inactivated by the tissue culture system. These findings suggest that the anatomical distribution of AHF antigen in vascular endothelial cells may play a crucial role in hemostasis, and cultured endothelial cells may be a valuable model for understanding AHF synthesis and release.The study investigates the synthesis of antihemophilic factor (AHF, Factor VIII) antigen by cultured human endothelial cells. Immunofluorescence studies using monospecific rabbit antibody to human AHF confirmed the presence of AHF antigen in endothelial cells, while control studies with smooth muscle cells and fibroblasts were negative. Radioimmunoassay demonstrated that cultured endothelial cells contain AHF antigen, which is released into the culture medium. Endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions from the culture media. The incorporation of radioactivity into immunoprecipitable protein was specific for AHF antigen, as it was not detected in smooth muscle cell media. However, despite the active synthesis of AHF antigen, no procoagulant activity was detected in the culture medium. Experiments using cycloheximide inhibited protein synthesis and [³H]leucine incorporation, but AHF antigen synthesis resumed after the inhibitor was removed. The study also found that exogenous AHF procoagulant activity was not inactivated by the tissue culture system. These findings suggest that the anatomical distribution of AHF antigen in vascular endothelial cells may play a crucial role in hemostasis, and cultured endothelial cells may be a valuable model for understanding AHF synthesis and release.