The relation between intracellular and extracellular [Na+] and [Ca2+] and membrane potential during stimulation of non-N-methyl-D-aspartate glutamate receptors has been studied in cerebellar granule cells using the fluorescent indicators SBFI, fura-2 and the bisoxonol membrane potential probe DiBaC4(3). Kainate increased both [Ca2+]i (intracellular [Ca2+]) and [Na+]i (intracellular [Na+]) and depolarized the membrane. This elevation of [Ca2+]i was only partially dependent on the presence of extracellular Na+ at the time of kainate addition. Removal of extracellular Na+ itself had a very minor effect on the [Ca2+]i or membrane potential of unstimulated cells. If extracellular Na+ was removed (in order to reverse the [Na+] gradient) or its concentration reduced during stimulation with kainate, the membrane depolarization recovered as expected. However, the intracellular level of sodium recovered only very slowly and the [Ca2+]i rose sharply, rather than recovering as might be expected on repolarization of depolarized cells possessing voltage sensitive calcium channels. This effect of extracellular [Na+] reduction on [Ca2+]i was mimicked by ouabain, another agent that causes accumulation of [Na+] in cells. These results suggest that Na+/Ca2+ exchange may play a major role in calcium homeostasis in stimulated cells, and that the levels of Na+ inside and outside the cell are critical in determining the effect of receptor stimulation on the intracellular [Ca2+].