Mechanisms that regulate endothelin (ET) in the perinatal lung are complex and poorly understood, especially with regard to the role of ET before and after birth. We hypothesized that the ET system is developmentally regulated and that the balance of ET_A and ET_B receptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and adult sheep. Lung preproET-1 mRNA levels, tissue ET peptide levels, and cellular localization of ET-1 expression were determined by Northern blot analysis, peptide assay, and immunohistochemistry in distal lung tissue from fetal lambs between 70 and 140 days (term = 145 days), newborn lambs, and ewes. Lung mRNA expression for the ET_A and ET_B receptors was also measured at these ages. We found that preproET-1 mRNA expression increased from 113 to 130 days gestation. Whole lung ET protein content was highest at 130 days gestation but decreased before birth in the fetal lamb lung. Immunolocalization of ET-1 protein showed expression of ET-1 in the vasculature and bronchial epithelium at all gestational ages. ET_A receptor mRNA expression and ET_B receptor mRNA increased from 90 to 125 and 135 days gestation. To determine changes in activity of the ET_A and ET_B receptors, we studied the effect of selective antagonists to the ET_A or ET_B receptors at 120, 130, and 140 days of fetal gestation. ET_A receptor-mediated vasoconstriction increased from 120 to 140 days, whereas blockade of the ET_B receptor did not change basal fetal pulmonary vascular tone at any age examined. We conclude that the ET system is developmentally regulated and that the increase in ET_A receptor gene expression correlates with the onset of the vasodilator response to ET_A receptor blockade. Although ET_B receptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions. We speculate that changes in ET receptor activity play important roles in regulation of pulmonary vascular tone in the ovine fetus.