Thrombin stimulates prostacyclin formation in cultured human endothelial cells. However, a countervailing process that prevents prostacyclin overproduction has not been described previously. In this study, we demonstrate that Factor Xa can inhibit prostacyclin synthesis induced by thrombin or sodium arachidonate. The required concentration of Factor Xa represents activation of only 2% of the Factor X in plasma. The inhibition is reversed by a human monoclonal antibody directed against the light chain of Factor Va (Mr = 78,000), which suggests that Factor Va is required for this down-regulation of prostacyclin production. Confluent human endothelial cells (10(7)) contained 1.4 to 2.2 micrograms of Factor V antigen as measured by a competitive enzyme-linked immunosorbent assay. The results indicate that in endothelial cells Factor Xa may play a regulatory role in prostacyclin formation through interaction with Factor Va.