1. Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (DeltaPsim) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction. 2. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 microM glutamate had different consequences for DeltaPsim. KCl caused a small transient loss of DeltaPsim but in response to glutamate there was a profound loss of DeltaPsim. In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in DeltaPsim. 3. Using fura-2 to measure [Ca2+]c, responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura-2FF revealed a difference in the [Ca2+]c responses to KCl and glutamate, which clearly correlated with the loss of DeltaPsim. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio. 4. The glutamate-induced loss of DeltaPsim was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of DeltaPsim. Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+]c, either in response to KCl or to glutamate in cultures at 7-10 DIV. 5. In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+]c. These data suggest that [Ca2+]c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+]c homeostasis during glutamate toxicity.