To investigate the effects of hyperoxia on mitochondrial multienzyme complex III (cytochrome, Cytb) and V (ATPase6, 8) in premature newborn rat lung, the 1-day-old preterm SD rats were randomly assigned to hyperoxia group and air group. The rats in hyperoxia group were continuously exposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day(s) of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6, 8 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blotting was used to detect the expression of Cytb protein in lung tissue. The results showed that compared with air group, Cytb mRNA expression was significantly increased (P>0.05) after 1, 4 day(s) of exposure. The general tendency decreased after 7 days, and its expression became weak but difference in mRNA expression between the two groups was not significant (P>0.05). ATPase6 mRNA expression was significantly increased 1 day after the exposure (P<0.05) and did not show any significant change 4, 7, 10 days after the exposure (P>0.05). At the 14th day, ATPase6 mRNA expression was significantly increased (P<0.05). ATPase8 mRNA expression did not show any significant change 1, 4, 10 day(s) after the exposure (P>0.05). At the 7th and 14th day, ATPase8 mRNA expression was significantly increased (P<0.05). Western blotting showed that Cytb protein expression was increased 1,4 day(s) after the exposure, but the difference between the two groups was not significant (P>0.05). The general tendency was decreased after 7 days, and its expression became weak but difference was not significant 7, 10 days after the exposure (P>0.05). At day 14 its expression became significantly weak (P<0.05). We are led to conclude that exposure to high concentrations of oxygen can significantly change the expression of Cytb and ATPase6, 8, which results in uncoupling of oxidative phosphorylation in mitochondrial respiration chain, and plays an important role in the mechanism of hyperoxia-induced lung injury.