The concept of the mitochondrial permeability transition (mPT) has been used to explain cell death induced by calcium deregulation, which is in turn induced by a disruption in the mitochondrial loading capacity of cytosolic calcium (CLC). Whether mitochondria have specific morphologies representing the CLC and the mPT remains controversial. We examined ultrastructural changes in the mitochondria of cultured hippocampal neurons preconditioned with oxygen-glucose deprivation (OGD) for 30 min (30OGD) or 120 min (120OGD). The CLC was then evaluated using simultaneous imaging of the mitochondrial and plasma Ca++ concentrations after the induction of Ca++ influx by the application of glutamate. In the 30OGD group, the CLC increased as the mitochondria rapidly reacted to the increase in plasma Ca++, which was soon cleared. In the 120OGD group, however, the CLC was disturbed because the mitochondrial uptake of Ca was blunted, and the plasma Ca++ was not cleared after glutamate application. We classified the specific morphological changes in the mitochondria according to a previously reported classification. Rounded mitochondria with scarce interior content were observed in the 120OGD group, a model of prolonged lethal OGD, and disruptions in the mitochondrial outer membrane were frequently confirmed, suggesting mPT. The 30OGD group, a model of enhanced CLC in preconditioned neurons, was characterized by round mitochondria with condensed matrices. After glutamate application, the mitochondria became even more rounded with expanded matrices, and outer membrane disruptions were occasionally seen. Our observations suggest that subpopulations of mitochondria with specific morphologies are linked to the CLC and mPT.