The effect of hyperoxia alone and in combination with inhaled nitric oxide on the integrity of lung mtDNA in vivo was evaluated in F344 rats. PCR amplification of lung mtDNA using two sets of primers that span 10.1kb of the mtDNA revealed that inhalation of 20 ppm nitric oxide in conjunction with hyperoxia (>95% O₂) reduced the amplification of the mtDNA templates by 10±1% and 26±3% after 24 hours of exposure. The reduction in mtDNA amplification was sustained after 48 hours of exposure with decreases of 7±2 % to 23±3% respectively for the two probes. The ability of mtDNA to amplify was not compromised in rats exposed to sublethal hyperoxia (80% O₂) even in the presence of 20 ppm inhaled nitric oxide. Surprisingly, exposure to >95% O₂ alone for either 24 or 48 h did not compromise the integrity of mtDNA templates as compared to air exposed controls despite evidence of genomic DNA injury. Interestingly, inhaling nitric oxide alone for 48 hours increased mtDNA amplification by 12±2% to 21±7%. The injury to the lung mitochondria DNA after exposure to >95% O₂ plus 20 ppm nitric oxide was transient as rats exposed to the two gases for 24 h and allowed to recover in room air displayed increased amplification, with levels exceeding controls by 20±3% to 29±4%. The increased amplification was not due to cellular proliferation as immunohistochemistry for the proliferation antigen Ki67 failed to show any significant cellular proliferation following exposure and recovery. The increased amplification was not due to increased mitochondrial number or increased mtDNA as the ratio of pulmonary mtDNA to genomic DNA remained the same between treatment groups. The results indicate that hyperoxia fails to induce significant injury to mtDNA and while inhalation of nitric oxide with hyperoxia results in mtDNA damage the lesions are rapidly repaired during recovery.