Background— Endothelin-1 (ET-1) has been implicated in the pathophysiology of pulmonary hypertension. In 1-month-old lambs with increased pulmonary blood flow, we have demonstrated early alterations in the ET-1 cascade. The objective of this study was to investigate the role of potential later alterations of the ET cascade in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow. Methods and Results— Eighteen fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 8 weeks after spontaneous delivery. Compared with age-matched control lambs, lung tissue ET-1 levels were increased in shunt lambs (317.2±113.8 versus 209.8±61.8 pg/g, P <0.05). In shunt lambs (n=9), exogenous ET-1 induced potent pulmonary vasoconstriction, which was blocked by the ET _A receptor antagonist PD 156707 (n=3). This pulmonary vasoconstriction was mimicked by exogenous Ala ^1,3,11,15 ET-1 (4 Ala ET-1), the ET _B receptor agonist, and was blocked by the ET _B receptor antagonist BQ 788 (n=3). However, in control lambs (n=7), ET-1 and 4 Ala ET-1 did not change pulmonary vascular tone. In contrast to 4-week-old shunt lambs, immunohistochemistry revealed the emergence of ET _B receptors on smooth muscle cells in the vasculature of 8-week-old shunt lambs. Conclusions— Over time, increased pulmonary blood flow and/or pressure results in the emergence of ET _B -mediated vasoconstriction, which coincides with the emergence of ET _B receptors on smooth muscle cells. These data suggest an important role for ET _B receptors in the pathophysiology of pulmonary hypertension in this animal model of increased pulmonary blood flow.