The purpose of our work was to study the relationship between glutamate (GLU)-induced mitochondrial depolarization and deterioration of neuronal Ca2+ homeostasis following a prolonged GLU challenge. The experiments were performed on cultured rat cerebellar granule cells using the fluorescent probes, rhodamine 123 and fura-2. All the cells, in which 100 microM GLU (10 microM glycine, 0 Mg2+) induced only relatively slight mitochondrial depolarization (1.1-1.3-fold increase in rhodamine 123 fluorescence), retained their ability to recover [Ca2+]i following a prolonged GLU challenge. In contrast, the cells in which GLU treatment induced pronounced mitochondrial depolarization (2-4-fold increase in rhodamine 123 fluorescence), exhibited a high Ca2+ plateau in the post-glutamate period. Application of 3-5 mM NaCN or 0.25-1 microM FCCP during this Ca2+ plateau phase usually failed to produce a further noticeable increase in [Ca2+]i. Regression analysis revealed a good correlation (r2 = 0.88 +/- 0.03, n = 19) between the increase in the percentage of rhodamine 123 fluorescence and the post-glutamate [Ca2+]i. Collectively, the results obtained led us to conclude that the GLU-induced neuronal Ca2+ overload was due to the collapse of the mitochondrial potential and subsequent ATP depletion.