Nitric oxide (NO), produced by the enzyme endothelial nitric oxide synthase(eNOS), modulates basal pulmonary vascular tone in the fetus and contributes to the fall in pulmonary vascular resistance at birth. eNOS is developmentally regulated in fetal rats, with gene expression peaking just prior to term and falling postnatally. A similar pattern is suggested in fetal lambs by physiologic studies which demonstrate increasing endothelium-dependent vasodilation during late gestation which peaks at term. However, whether developmental changes in eNOS expression account for these physiologic changes is not known. Therefore, to study the ontogeny of eNOS expression, we measured eNOS protein, activity, and mRNA in lung tissue of fetal lambs. We studied whole lung samples from 57 animals, which were divided into 8 fetal groups(70, 90, 113, 118, 125, 130, 135, and 140 d gestation; term=147 d), 3 neonatal groups (1, 5, and 17 d), and postpartum ewes. Western blot analysis was performed using a monoclonal antibody against eNOS and each individual blot was normalized to an endothelial cell lysate standard. eNOS activity was determined using the L-arginine-to- citrulline conversion assay and results were normalized to protein concentration. Northern blot analysis was performed on total RNA from whole lung using a cDNA probe for eNOS. Prenatally, eNOS protein, activity, and mRNA peak at 113-118 d gestation (p<0.05 vs. 70, 130, 135, and 140 d groups). Postnatally, a second peak in eNOS mRNA levels occurs at 1 d (p<0.05 vs. 5 and 17day lamb and postpartum ewe), and protein peaks at 5 d (p<0.05 vs. 1 and 17 day lambs and postpartum ewe). However, no postnatal peak in eNOS activity is observed. We conclude that eNOS expression peaks at ≈80% of term in the lungs of fetal lambs and falls thereafter. The physiologic significance of the early peak of eNOS expression in the lung is uncertain, but its timing coincides with the onset of a rapid increase in fetal lung growth. We speculate that eNOS may contribute to the rapid growth and development of the lung circulation at this gestational age and that factors independent of eNOS expression modulate the near-term increase in NO-dependent vasodilation.