Free radical generation in the neonatal mouse brain subjected to acute hypoxia was measured directly by using electron spin resonance spectroscopy (ESR). Free radical density, an index of free radical content, was investigated during exposure to nitrogen gas (N2 group) or carbon dioxide gas (CO2 group) of high purity, and its subsequent recovery in air. Free radical density in the N2 group declined during hypoxia, increased over the control level at 10 min of recovery, and then returned to the prehypoxic level. In the CO2 group, it increased during hypoxia, reached the maximum level at 20 min of recovery, and then returned to the control level. The main origin of the spectrum in control brain was considered as being the coenzyme Q10 (CoQ10) radical in the mitochondria. The change of free radical density during hypoxia and recovery in the N2 group and that during recovery in the CO2 group was thought to be correlated with changes in CoQ10 radicals indicating mitochondrial function. The increase of free radical density in the CO2 group during hypoxia suggested the generation of free radicals other than the CoQ10 radical. We conclude that the free radicals which appeared during CO2 hypoxia may play a role in producing the differences in brain injury between the two kinds of hypoxia.