Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices this triggers a massive depolarization of CA1 neurons, and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by FCCP (1 µM) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 µg/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 µM), diphenyleneiodonium (25 µM) or antimycin A (20 µM) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 µM) hastened HSD onset and increased HSD duration. At higher concentrations cyanide (1 mM), azide (2 mM), and FCCP (10 µM) triggered SD episodes on their own. Compared to control HSD the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH and FAD autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH or FAD levels and the time to HSD onset. Therefore, ATP depletion or a cytosolic reducing shift due to NADH/FADH₂ accumulation cannot serve as a general explanation for the hastening of HSD onset upon mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.