Mechanical ventilation (MV) is associated with diaphragmatic oxidative stress that contributes to both diaphragmatic atrophy and contractile dysfunction. However, the pathways responsible for oxidant production in the diaphragm during MV remain unknown. To address this issue, we tested the hypothesis that nitric oxide synthase (NOS) activity is elevated during MV resulting in nitration of diaphragmatic proteins. Rats were mechanically ventilated for 18 hours and time matched, anesthetized, but spontaneously breathing animals served as controls. Protein levels of eNOS, iNOS, and nNOS were measured in diaphragms from all animals. 3-nitrotyrosine levels were also measured as an index of protein nitration and S-nitrosothiol levels were measured as a marker of nitric oxide reactions with molecules containing sulfhydryl groups. Levels of nitrates and nitrites were measured as markers of stable end products of nitric oxide metabolism. Finally, as a marker of oxidative stress, diaphragmatic levels of reduced glutathione (GSH) were also analyzed. MV did not promote an increase in diaphragmatic protein levels of eNOS or nNOS. Moreover, iNOS was not detected in either experimental group. Consistent with these findings, MV did not elevate diaphragmatic 3-nitrotyrosine levels in any sub-cellular fraction of the diaphragm including the cytosolic, mitochondrial, membrane, and insoluble protein fractions. Moreover, prolonged MV did not elevate diaphragmatic levels of S-nitrosothiols, nitrate, or nitrite. Finally, prolonged MV significantly reduced diaphragmatic levels of GSH which is consistent with diaphragmatic oxidative stress. Collectively, these data reveal that MV-induced oxidative stress in the diaphragm is not due to increases in nitric oxide production by NOS.