In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca(2+) ([Ca(2+)](c)) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca(2+)influx. The [Ca(2+)](c) level increased sharply, followed by a rapid increase in mitochondrial Ca(2+) [Ca(2+)](m). The increase in the [Ca(2+)](m) level accompanied a reduction in the [Ca(2+)](c) level. After reaching a peak, the [Ca(2+)](c) level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.