Extrinsic pathway inhibitor (EPI) is a potent inhibitor of the factor VIIa-tissue thromboplastin complex. The effect of heparin on EPI activity was studied using a chromogenic substrate assay. Addition of heparin to test plasma or whole blood in vitro increased EPI activity. This increase in EPI activity was reduced by the addition of polybrene and/or antibodies to antithrombin (anti-AT). Polybrene was therefore added to the assay system. However, part of this effect (up to 20% of baseline value) was not abolished. After intravenous injection, EPI activity increased dose-dependently. The increase was about 200% of baseline value after 7500 U heparin, and was not reduced by the addition of polybrene and/or anti-AT. A slower and prolonged increase in EPI activity occurred after subcutaneous injections of unfractionated heparin and low molecular weight heparin (LMWH). Venous occlusion failed to increase EPI activity levels. Normal EPI values were observed in patients with severe liver cirrhosis and during warfarin treatment. Gel filtration of human endothelial cell culture supernatant revealed one inhibitory fraction with molecular weight about 43.000. We conclude that EPI probably is produced in endothelial cells and may be released by heparin.