Cellular injury was evaluated in superfused cerebrocortical slices (350 µm) from 7-day-old Sprague–Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.