The effect of hyperoxia alone and in combination with inhaled nitric oxide (NO) on the integrity of lung mitochondrial DNA (mtDNA) in vivo was evaluated in Fischer 344 rats. PCR amplification of lung mtDNA using two sets of primers spanning 10.1 kb of the mtDNA revealed that inhalation of 20 ppm of NO in conjunction with hyperoxia (>95% O2) reduced the amplification of mtDNA templates by 10 +/- 1% and 26 +/- 3% after 24 h of exposure. The ability of mtDNA to amplify was not compromised in rats exposed to 80% O2, even in the presence of 20 ppm of inhaled NO. Surprisingly, exposure to >95% O2 alone for either 24 or 48 h did not compromise the integrity of mtDNA templates compared with air-exposed controls, despite evidence of genomic DNA injury. Interestingly, inhaling NO alone for 48 h increased mtDNA amplification by 12 +/- 2% to 21 +/- 7%. Injury to the lung mtDNA after exposure to >95% O2 plus 20 ppm of NO was transient as rats allowed to recover in room air after exposure displayed increased amplification, with levels exceeding controls by 20 +/- 3% to 29 +/- 4%. Increased amplification was not due to cellular proliferation or increased mitochondrial number. Moreover, 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 whereas inhalation of NO with hyperoxia results in mtDNA damage, the lesions are rapidly repaired during recovery.