To better understand the mechanisms whereby pulmonary blood flow regulates pulmonary vascular bed growth in congenital heart lesions, 42 day old Sprague-Dawley rats (n = 24) had creation of an aortocaval shunt to increase pulmonary blood flow for six weeks. The shunt resulted in a significant increase in heart (2.89 ± 0.4 vs 6.1 ± 0.9, P ≤ 0.05) and lung (1.93 ± 0.5 vs 4.6 ± 1.4, P ≤ 0.05) to body weight (sham vs shunt, mean ± standard deviation) without significantly altering pulmonary (16 ± 3 vs 19 ± 4, NS) or carotid (125 ± 5 vs 130 ± 6, NS) blood pressures (mean mm Hg ± standard deviation). Histologic examination of the lungs revealed significant thickening of the pulmonary arterial medial wall relative to the vessel diameter (35% ± 7.2 vs 57% ± 11.8, P ≤ 0.05, sham vs shunt, mean ± standard deviation) and increased muscularization of very small arteries (≤ 80μM) as evidenced by α-actin smooth muscle staining. Proliferating cell nuclear antigen staining and bromo-deoxyuridine labeling of proliferating cells demonstrated that the thickened arterial wall was a result of medial hypertrophy and not proliferation. To determine the role of nitric oxide (NO) in the regulation of the arteriolar hypertrophy or the affect of hypertrophy on NO expression in the pulmonary vasculature, endothelial NO synthase (eNOS) gene and protein expression were determined in lungs from shunt and sham operated animals. Western and northern analysis demonstrated that eNOS protein and mRNA levels were not altered in the shunt lungs. In addition NAPDH diaphorase staining was negative for increased NOS in the hypertrophied pulmonary vasculature of the shunt animals. Therefore increased pulmonary flow without increased pressure resulted in pulmonary artery medial hypertrophy and not hyperplasia. We speculate that the pulmonary vascular remodeling resulting from increased pulmonary blood flow alone is not mediated by eNOS.