The endothelial cells lining the vessel wall can modulate vasomotor tone by releasing vasoactive factors, such as endothelial-derived constricting factors. We observed that alpha-thrombin, but not catalytically inactivated alpha-thrombin, mediated the release of two pulmonary vasoconstrictor peptides into the venous effluent of guinea pig lungs. These peptides elicited a slow-onset, long-lasting pulmonary vasoconstriction similar to the effect of endothelin, an endothelial-derived 21-amino acid vasoconstrictor peptide previously isolated from cells in culture. One of the isolated peptides coelutes with endothelin upon reverse-phase HPLC with an acetonitrile gradient and has a molecular weight comparable to endothelin as determined by gel-permeation HPLC. The other vasoconstrictor peptide elutes earlier than endothelin on reverse-phase HPLC and exhibits a lower molecular weight. The studies show the release of endothelin-like pulmonary vasoconstrictor peptides in the intact lung by alpha-thrombin, a central regulatory enzyme in hemostasis.