Hyperoxia (HP) exposure inducts reactive oxygen species (ROS) in the lungs that may result in lung injury, including alveolar epithelial and endothelial cells. Lactate dehydrogenase (LDH) activity relates to glycolysis, whereas glutathione peroxidase (Gpx) activity relies on the pentose phosphate pathway (PPP). The purpose of this study was to examine early ROS-induced alveolar pathological changes in relation to the activity of glutathione peroxidase (GPx) and lactate dehydrogenase (LDH) activity. Twenty adult male rats, matched with age and body weight, were randomly assigned to two groups, control and experimental. The experimental group was exposed to hyperoxia for 24 h. Ultrastructure examination showed degenerated pneumocyte type I, containing swollen mitochondria associated with dilated rough endoplasmic reticulum, and was projecting into the alveolar lumen. Pneumocyte II showed mitochondria swelling and hyperplasia and was desquamated in structure, depleted in surfactant, and falling into the alveolar lumen. Pulmonary capillary showed distention without observed damage in the endothelial layer. Following HP, the average (+/-) free radical (FR) production increased significantly (p<.05) from the baseline control of 181.20+/-30.06 to 260.30+/-68.10 (Carr U) and average (+/-SD) GPx activity also increased significantly (p<.05) from the baseline control of 8178.30+/-2402.62 to 19,589.50+/-2392.44 (U/L), whereas average (+/-SD) LDH activity decreased significantly (p<.05) from baseline control of 194.11+/-75.52 to 42.68+/-11.41 (U/L), which demonstrated slowing down of glycolysis. Based on these results it can be concluded that exposure to high inspired oxygen inducted the buildup of mitochondria-driven ROS that was related to early injury in the alveolar epithelium without obvious endothelium injury.