Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relation, isotonic fatigability and nitrotyrosine levels were assessed under hypoxic (Po₂ ~ 6.5 kPa) and hyperoxic (Po₂ ~ 88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min) and the NOS inhibitor L-NMMA (90 min). Hypoxiainduced downward shift of the force-frequency relation was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels, but did not affect force generation. Strenuous contractions impaired force generation, but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relation was found between diaphragm nitrotyrosine formation and in vitro force generation.