Utilizing Indo-1 microfluorimetry, we have investigated the role of mitochondria and Na+/Ca2+ exchange in buffering calcium loads induced by glutamate stimulation or depolarization of cultured rat forebrain neurons. A 15 sec pulse of 3 microM glutamate or 50 mM potassium with veratridine was followed by a 2 min wash with a solution containing either Na(+)-free buffer or the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or both. For glutamate-induced Ca2+ loads, a Na(+)-free wash delayed recovery to baseline by twofold, mitochondrial uncoupling delayed recovery by greater than fourfold, and the combined treatment essentially prevented recovery of [Ca2+]i for the duration of the wash. Although the depolarization stimulus was able to elicit a larger peak [Ca2+]i, the neurons required significantly less time to recover from depolarization-induced Ca2+ loads after identical wash manipulations, indicating a fundamental difference between calcium loads induced by glutamate as opposed to those induced by depolarization. We show evidence that the delayed recovery is not primarily the result of perturbations in intracellular pH regulation and have also demonstrated that a substantial portion of the delayed recovery is independent of Ca2+ entry during the washout phase. We conclude that glutamate and depolarization both induce Ca2+ loads whose buffering is critically dependent on functional mitochondria and secondarily reliant on Na+/Ca2+ exchange. The two systems overlap and seem to be responsible for buffering most of the glutamate-induced Ca2+ load, because manipulations that compromised both systems completely disabled the neurons' ability to recover [Ca2+]i to baseline.