Pathological activation of glutamate receptors with consequent disturbances in Ca²⁺ homeostasis and collapse of mitochondrial potential plays a major role in the neuronal death that follows episodes of anoxia or ischaemia. We have been concerned to understand the relationships between changes in mitochondrial membrane potential (Δψm) and the failure of cytoplasmic calcium homeostasis following exposure of rat hippocampal neurons to toxic concentrations of glutamate. We have used fluorescence imaging to measure Δψm and [Ca²⁺]_c using Rhodamine123 and Fura-FF respectively in primary cultures of hippocampal neurons (10-15 days in vivo). Application of glutamate (100μM) caused a stereotypical response consisting of a delayed loss of Δψm coincident with a secondary increase in [Ca²⁺]_c. For a period of about 10 min, these responses were reversed by removal of external Ca²⁺, but after this period, removal of external Ca²⁺ had no effect on either variable. The mitochondrial depolarization but not the secondary increase in [Ca²⁺]_c was blocked by Ru360 (20 μM), an inhibitor of mitochondrial Ca²⁺ uptake. After prolonged glutamate exposure (10-15 min), when the [Ca²⁺]_c response had progressed to an irreversible phase and mitochondrial depolarization was complete in most cells, reduction of [Ca²⁺]_c using the membrane permeant calcium chelator EGTA-AM (50 μM) promoted recovery of Δψm in the majority of cells (n=169/208 for EGTA-AM), suggesting that the mitochondrial response was dependent on the sustained presence of calcium and was still reversible. Mitochondrial potential also recovered in response to TMPD and ascorbate, which provide electrons to mitochondrial complex IV, suggesting that the effect of calcium involves impaired substrate supply or respiratory chain activity. The mitochondrial substrates methyl-succinate (10 mM) and TMPD (200μM) plus ascorbate (5mM) recovered potential in the majority neurons (88%, n=79/90 for TMPD/ascorbate; 73%, n=92/126 for Me-succinate) in this period after glutamate addition. In thapsigargin-treated neurons 5μM ionomycin in a Ca²⁺-free saline raised [Ca²⁺]_c, almost certainly releasing mitochondrial calcium. Furthermore, permeabilisation of the cells with digitonin (10 μM) after 10-15 min glutamate exposure also showed that mitochondria were still intact and were still responsive to additions of calcium and substrates. Thus, after 10-15 min of glutamate exposure mitochondria in hippocampal neurons are still intact, maintain a high calcium content and are able to recover a potential. The loss of potential has been attributed to opening of the permeability transition pore (mPTP) which would lead to loss of mitochondrial calcium and a depolarization that could not be reversed by substrate. We conclude that the early profound mitochondrial depolarization in neurons during excitotoxic glutamate exposure cannot be due to mPTP opening.