W e examined the effect of bovine aortic endothelial cell culture supernatants upon the generation of procoagulant activity by human blood monocytes. Confluent endothelial monolayers were cultured for up to 96 h. At timed intervals, culture supernatants were collected and incubated for 5 h with lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The procoagulant activity of mononuclear cell lysates was determined in a one-stage clotting assay. In five experiments, procoagulant activity with culture supernatant (time 0) was 2,294 +/- 761 U/ml (mean +/- SEM). Culture supernatants from endothelial cells incubated for 24-96 h strongly inhibited mononuclear cell generation of procoagulant activity. Indomethacin (10 microM) added to endothelial cells delayed the appearance of procoagulant inhibitor for 72 h. Bovine aortic smooth muscle cell culture supernatants did not inhibit procoagulant activity. The inhibitor was heat stable, effective at 1:50 dilution, soluble, and acid sensitive, with a molecular weight of less than 1,500. Further studies on subpopulations of mononuclear cells demonstrated that endothelial inhibitor selectively decreased the generation of monocyte procoagulant activity and interfered with T lymphocyte amplification of monocyte production of procoagulant activity. Thus, we have demonstrated that endothelial cells elaborate a potent inhibitor of monocyte procoagulant activity.