Objective: Previous studies in fetal lamb have suggested that chronic pulmonary hypertension in utero leads to decreased expression of endothelial nitric oxide synthase (eNOS) in pulmonary arteries. However, the role of decreased eNOS activity in persistent pulmonary hypertension of the newborn (PPHN) is unknown. We proposed the hypothesis that umbilical vein endothelial cells cultured from infants who subsequently developed PPHN will have decreased eNOS expression. Study Design: Umbilical cords from meconium stained infants were collected in phosphate buffered saline and stored at 4°C. Endothelial cells were cultured from umbilical vein of 6 infants who subsequently developed PPHN. Cells were also harvested from nine umbilical cords of normal term infants. Total RNA was isolated from confluent cultures of vascular endothelial cells. Messenger RNA was reverse transcribed using a specific human eNOS anti-sense primer. PCR was performed for 30 cycles with a denaturation temp of 94°C for 45 sec, an annealing temp of 53°C for 45 sec and a extension temp of 72°C for 60 sec. PCR products were visualized by agarose gel electrophoresis. Results: PCR amplification revealed an expected band of approximately 550 bp in all nine controls. Among patients with PPHN, four had no detectable eNOS transcription and two did. Amplification of control β-actin cDNA was noted among all patient and control samples. Conversely, no amplification was present in any negative controls to which no cDNA was added. There was no difference noted in the course and outcome of patients who had or did not have the eNOS band. However, there was an acidotic initial arterial blood gas (pH 7.19- 7.29) in patients who did not show the eNOS band. Conclusion: Endothelial NOS mRNA was detected in all normal term infants in this study. However, four out of 6 infants with PPHN did not express eNOS mRNA. The development of PPHN is multifactorial and a decrease in the expression of the eNOS mRNA may play a role in some infants. Whether the decreased expression of eNOS is a cause of PPHN or a result of intrapartum stress needs to be studied.