Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.