Alterations in brain cell membrane structure and function during cerebral hypoxia were investigated by measuring Na+, K+-ATPase activity and levels of lipid peroxidation products in brain cell membranes obtained from newborn piglets following exposure to 60 min of hypoxic hypoxia in vivo. Cerebral hypoxia was documented as a decrease in the ratio of phosphocreatine to inorganic phosphate (PCr/Pi) using 31P-NMR spectroscopy. During hypoxia (FiO2 0.07-0.11), PCr/Pi decreased 28–47% compared to the corresponding baseline value without a decrease in cerebral ATP levels. No change in brain cell membrane Na+, K+-ATPase activity was observed for changes in PCr/Pi of < 30%. When PCr/Pi was at least 30% lower than baseline, Na+, K+-ATPase activity decreased linearly as a function of the decrease in PCr/Pi (r = 0.95). Levels of lipid peroxidation products (conjugated dienes and fluorescent compounds) increased significantly as PCr/Pi decreased. These data suggest that below a critical threshold value of oxidative metabolism there are progressive changes in brain cell membrane structure and function during cerebral hypoxia, and demonstrate that membrane alterations occur prior to changes in cellular ATP levels.