This study examines the causes of hypothermia and rewarming injury in CA1, CA3, and dentate neurons in rat hippocampal slice cultures. Neuronal death, assessed with propidium iodide or Sytox® fluorescence, Fluoro-Jade labeling, and Cresyl Violet staining, depended on the severity and duration of hypothermia. More than 6 h at temperatures less than 12 °C followed by rewarming to 37 °C (profound hypothermia and rewarming, PH/RW) caused swelling and death in large number of neurons in CA1, CA3, and dentate. During PH, [ATP] decreased and [Ca2+]i and extracellular [glutamate] increased, with neuron rupture and nuclear condensation following RW. The data support the hypothesis that neuronal death from PH/RW is excitotoxic, due to ATP loss, glutamate receptor activation and Ca2+ influx. We found that antagonism of N-methyl-D-aspartate (NMDA) receptors, but not 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid or metabotropic glutamate receptors, decreased neuron death and prevented increases in [Ca2+]I caused by PH/RW. Chelating extracellular Ca2+ decreased PH/RW injury, but inhibiting L- and T-type voltage-gated Ca2+ channels, K+ channels, Ca2+ release from the endoplasmic reticulum, and reverse Na+/Ca2+ exchange did not affect the Ca2+ changes or cell death. We conclude that the mechanism of PH/RW neuronal injury in hippocampal slices primarily involves intracellular Ca2+ accumulation mediated by NMDA receptors that activates necrotic, but not apoptotic processes.