Although the liver is known to be the major site of FVIII production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extra-hepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. As physiological stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it following treatment with agonists. Microvascular endothelial cells from lung, heart, intestine and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it following treatment with phorbol-myristate acetate (PMA) and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and vein did not constitutively secrete FVIII nor release it after treatment with agonists, likely due to a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions.