Spreading depression (SD) is a profound depolarization of neurons and glia that propagates in a wave-like manner across susceptible brain regions, and can develop during periods of compromised cellular energy such as ischemia, when it influences the severity of acute neuronal damage. Although SD has been well characterized in the cerebral cortex and hippocampus, little is known of this event in the Substantia Nigra (SN), a brainstem nucleus engaged in motor control and reward-related behavior. Transverse brain slices (250 ?m; P21-23 rats) containing the SN were subject to oxygen and glucose deprivation (OGD) tests, modeling brain ischemia. SD developed in lateral aspects of the SN within 3.3±0.2 min of OGD onset, and spread through the Substantia Nigra pars reticulata (SNr), as indicated by fast-occurring and propagating increased tissue light transmittance and negative shift of extracellular DC potential. These events were associated with profound mitochondrial membrane depolarization (??m) throughout the SN, as demonstrated by increased Rhodamine 123 fluorescence. Extracellular recordings from individual SNr neurons indicated rapid depolarization followed by depolarizing block, while dopaminergic neurons in the Substantia Nigra pars compacta (SNc) showed inhibition of firing associated with hyperpolarization. SD evoked in the SNr was similar to OGD-induced SD in the CA1 region in hippocampal slices. In the hippocampus, SD also developed during anoxia or aglycemia alone (associated with less profound ??m than OGD), while these conditions rarely led to SD in the SNr. Our results demonstrate that OGD consistently evokes SD in the SN, and that this phenomenon only involves the SNr. It remains to be established whether nigral SD contributes to neuronal damage associated with a sudden-onset form of Parkinson's disease known as 'vascular parkinsonism'.