The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca²⁺-permeable AMPA/kainate channels, which may permit rapid Ca²⁺ influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably forebrain GABAergic interneurons, consistently express large numbers of these channels but do not degenerate in ALS. Indeed, when subjected to identical excitotoxic exposures, motor neurons were more susceptible than GABAergic neurons to AMPA/kainate receptor-mediated neurotoxicity. Microfluorimetric studies were performed to examine the basis for the difference in vulnerability. First, AMPA or kainate exposures appeared to trigger substantial mitochondrial Ca²⁺ loading in motor neurons, as indicated by a sharp increase in intracellular Ca²⁺after addition of the mitochondrial uncoupler carbonyl cyanidep-(trifluoromethoxy)phenyl hydrazone (FCCP) after the agonist exposure. The same exposures caused little mitochondrial Ca²⁺ accumulation in GABAergic cortical neurons. Subsequent experiments examined other measures of mitochondrial function to compare sequelae of AMPA/kainate receptor activation between these populations. Brief exposure to either AMPA or kainate caused mitochondrial depolarization, assessed using tetramethylrhodamine ethylester, and reactive oxygen species (ROS) generation, assessed using hydroethidine, in motor neurons. However, these effects were only seen in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally, addition of either antioxidants or toxins (FCCP or CN⁻) that block mitochondrial Ca²⁺ uptake attenuated AMPA/kainate receptor-mediated motor neuron injury, suggesting that the mitochondrial Ca²⁺ uptake and consequent ROS generation are central to the injury process.