In the present study we tested the hypothesis that the cyclophilin D-dependent (CyD) mitochondrial permeability transition (CyD-mPT) plays an important role in glutamate-triggered delayed calcium deregulation (DCD) and excitotoxic neuronal death. We used cultured cortical neurons from wild-type C57BL/6 and cyclophilin D-knockout mice (Ppif(-/-)). Induction of the mPT was identified by following the rapid secondary acidification of mitochondrial matrices monitored with mitochondrially targeted pH-sensitive yellow fluorescent protein. Suppression of the CyD-mPT due to genetic CyD ablation deferred DCD and mitochondrial depolarization, and increased the survival rate after exposure of neurons to 10 microM glutamate, but not to 100 microM glutamate. Ca(2+) influx into Ppif(-/-) neurons was not diminished in comparison with WT neurons judging by (45)Ca accumulation. In both types of neurons, 100 microM glutamate produced greater Ca(2+) influx than 10 microM glutamate. We hypothesize that greater Ca(2+) influx produced by higher glutamate rapidly triggered the CyD-independent mPT in both WT and Ppif(-/-) neurons equalizing their responses to supra-physiologic excitotoxic insults. In neurons exposed to moderate but pathophysiologically-relevant glutamate concentrations, an induction of the CyD-mPT appears to play an important role in mitochondrial injury contributing to DCD and cell death.