Nitric oxide (NO), produced by the endothelial isoform of nitric oxide synthase (eNOS), is a critical mediator of pulmonary vascular structure and function in the developing lung, causing vasodilation and the inhibition of vascular smooth muscle growth. We have previously shown that pulmonary eNOS gene expression is developmentally regulated during late fetal and early postnatal life, suggesting the eNOS gene expression may be modulated by the state of pulmonary artery endothelial cell (PAEC) growth. We therefore studied the specific effects of cell growth on eNOS expression in cultured ovine fetal PAEC. To examine the effects of cell growth modified by two independent approaches, experiments were performed in cells at varying confluence, and in control cells versus cells stimulated with 20% ovine serum for 24-48 h. Cell proliferation was assessed by examining the incorporation of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into replicating DNA during thymidilate synthetase inhibition with 5-flouro-2′-deoxyuridine. BrdU-positive nuclei were identified by immunocytochemistry with a specific monoclonal antibody. eNOS expression was evaluated by determinations of cell lysate NOS enzymatic activity, which is a highly sensitive measure of changes in enzyme abundance. Compared to 100% confluent cells, cell proliferation was greater at 50% confluence (3.5±0.6% vs. 9.4±0.8% BrdU-positive nuclei, respectively). Greater cell proliferation was associated with attenuated NOS activity, which was 11.9±2.4 vs. 2.3±0.1 pmol/mg prot/min at 100% vs. 50% confluence, respectively. With serum stimulation, cell proliferation increased from 2.8±0.3% to 5.9±0.4% BrdU-positive nuclei. Paralleling the findings with varying confluence, NOS activity was attenuated in the more rapidly-proliferating cells, falling from 2.0±0.3 to 0.5±0.1 pmol/mg prot/min with serum stimulation. These findings contrast with those in systemic endothelium, which exhibit enhanced eNOS expression with greater cell growth. Thus, eNOS expression is markedly attenuated with the activation of cell growth in fetal PAEC. We postulate that eNOS downregulation in rapidly-growing PAEC may facilitate concomitant smooth muscle replication, thereby coordinating the growth of the two cell types during vascularization of the developing lung.