Inhaled nitric oxide (NO) has been used in the preoperative evaluation of patients with congenital heart disease and pulmonary hypertension. The purpose of this study was to characterize responses in pulmonary vascular resistance (PVR) to oxygen and increasing doses of NO during cardiac catheterization and to determine if any related factors affect the response of the pulmonary vascular bed to NO. A prospective analysis of 42 patients (median age, 3.0 years) with congenital heart disease and pulmonary hypertension who underwent NO testing was performed. Systemic vascular resistance (SVR) and PVR were assessed in room air, 100% oxygen, and oxygen plus 20, 40, and 80 parts per million (ppm) NO. Changes in pulmonary artery pressure, PVR, and SVR were assessed. The response to NO was then correlated to individual patient’s age, gender, type of heart defect, the presence of trisomy 21, and baseline PVR/SVR. There was a greater decrease in PVR and PVR/SVR with 20 ppm NO than with oxygen alone. There was no additional decrease at 40 or 80 ppm NO. There was no correlation between age, gender, type of congenital heart disease, and baseline PVR/SVR ratio with the degree of response to NO. Patients with trisomy 21 had less of a response to NO (p = 0.017) than patients without trisomy 21. There is no difference in determining PVR response with doses of NO beyond 20 ppm during cardiac catheterization. Age, gender, and baseline PVR/SVR ratio are not associated with responsiveness to NO. Patients with trisomy 21 may be less responsive to NO.