Pyramidal neurons in the CA1 region of the hippocampus are highly vulnerable to damage from hypoxia-ischemia, whereas neurons in the CA3 region and the dentate gyrus are more resistant. A similar pattern of vulnerability to loss of synaptic and membrane function occurs in the in vitro hippocampal slice preparation, suggesting that intrinsic factors are important in acute neuronal damage. Simultaneous recordings of DC potential and imaging of changes in light transmittance were made in slices from the middle one-third of the hippocampus to characterize the initiation and spread of depolarization and swelling during hypoxia-aglycemia. Hypoxic depolarization (HD) and associated optical changes were initiated simultaneously in the stratum oriens of the CA1 region and thereafter spread to the stratum radiatum of CA1 and later to the upper (inner) blade of the dentate gyrus. A decrease in light transmittance was associated consistently with depolarization in all regions (n = 22 slices). Investigation of the sequence of activation in intact slices showed that activation of the dentate gyrus arose independently of activation of the CA1 region. This was confirmed by recordings made from minislices in which CA1, CA3, and dentate regions were physically separated. HD and optical changes were never observed in the CA3 region, despite exposure to 40-60 min of combined hypoxia and aglycemia. In contrast, exposure to hypoxia after pretreatment of slices with altered tonicity or ion composition, which triggered episodes of spreading depolarization (SD), provoked depolarization and optical changes simultaneously in both CA1 and CA3 regions. Similarly, pretreatment with agents that cause severe metabolic impairment, such as dinitrophenol (DNP), also rendered the CA3 region vulnerable to subsequent hypoxia. This suggests that the CA3 region in hippocampal slices is normally resistant to HD and only becomes vulnerable after severe impairment of metabolic capacity. The similar order of vulnerability of in vitro and in vivo hippocampus to hypoxia-aglycemia supports the use of the hippocampal slice preparation to investigate early changes potentially contributing to hypoxic-ischemic injury.