Vascular endothelial cells produce endothelin-1, a peptide with potent vasoconstrictor and mitogenic properties. Heparin suppresses thrombin-stimulated endothelin-1 production in endothelial cells; this is consistent with its reported effect of lowering blood pressure. Since heparin is a heterogeneous mixture of glycosaminoglycans, we examined the effects of different fractions of heparin in suppressing endothelin-1 production in cultured endothelial cells. Heparin fractions differing in size and in antithrombin III affinity were prepared. The results show that the suppressive effect of heparin is independent of these properties of size and antithrombin III affinity. Heparin sulfate suppressed endothelin-1 production to a similar level as heparin. These experiments were conducted in a complete culture medium in the absence of added thrombin. To assess the role of endogenous thrombin in the medium on this process, we tested the effects of hirudin, a specific thrombin inhibitor peptide, on suppression of endothelin-1. Hirudin, like heparin, binds to the anion-binding exosite of thrombin. Hirudin alone, and combined with heparin, suppressed endothelin levels to the same extent as heparin. These experiments demonstrate that the suppressive effect of heparin is the result of its binding to the traces of thrombin in the culture medium, preventing stimulation of endothelin-1 production. This study supports the hypothesis that the functional thrombin receptor may participate in the stimulation of endothelin-1 by thrombin.