Life-threatening increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO), although the mechanisms remain unknown. In vitro data suggest that exogenous NO exposure inhibits endothelial NO synthase (NOS) activity. Thus the objectives of this study were to determine the effects of inhaled NO therapy and its acute withdrawal on endogenous NOS activity and gene expression in vivo in the intact lamb. Six 1-mo-old lambs were mechanically ventilated and instrumented to measure vascular pressures and left pulmonary blood flow. Inhaled NO (40 ppm) acutely decreased left pulmonary vascular resistance by 27.5 ± 4.7% (P < 0.05). This was associated with a 207% increase in plasma cGMP concentrations (P < 0.05). After 6 h of inhaled NO, NOS activity was reduced to 44.3 ± 5.9% of pre-NO values (P 0.05). After acute withdrawal of NO, pulmonary vascular resistance increased by 52.1 ± 11.6% (P < 0.05) and cGMP concentrations decreased. Both returned to pre-NO values within 60 min. One hour after NO withdrawal, NOS activity increased by 48.4 ± 19.1% to 70% of pre-NO values (P < 0.05). Western blot analysis revealed that endothelial NOS protein levels remained unchanged throughout the study period. These data suggest a role for decreased endogenous NOS activity in the rebound pulmonary hypertension noted after acute withdrawal of inhaled NO.