Introduction: Life-threatening increases in pulmonary vascular resistance (PVR) have been noted upon acute withdrawal of inhaled nitric oxide (iNO). However, the mechanisms of this rebound pulmonary hypertension are unknown. In vitro data suggests that exogenous NO exposure inhibits endothelial nitric oxide synthase (NOS) activity. The objective of this study was to determine the effects of iNO, and its acute withdrawal, on endogenous NOS activity and gene expression, in vivo, in intact lambs. Methods: Six one-month-old lambs were mechanically ventilated and instrumented to measure vascular pressures and left pulmonary blood flow, iNO (40 ppm) was administered for 24 hours and then acutely withdrawn. The hemodynamic variables were monitored continuously. Intermittently, lung biopsies were performed for NOS activity (using the conversion of 3H-arginine to 3H-citrulline) and Western blot analysis. Results: iNO decreased left PVR and NOS activity. Upon withdrawal of iNO, PVR acutely increased, and then decreased to pre-iNO values within one hour. Concurrently, NOS activity increased to 70% of pre-iNO values within one hour. ENOS protein expression remained unchanged throughout the study period. Conclusions: iNO reversibly inhibits endogenous NOS activity but not gene expression in the intact Iamb. These data suggest a role for decreased endogenous NOS activity in the rebound pulmonary hypertension noted upon acute withdrawal of iNO.