This study examined the effects of clinically relevant concentrations of isoflurane on the amplitude of NMDA receptor current (I-NMDA) and the expression of cytochrome C in cultured developing rat hippocampal neurons. The hippocampi were dissected from newborn Sprague-Dawley rats. Hippocampal neurons were primarily cultured for 5 days and then treated with different concentrations of isoflurane [(0.25, 0.5, 0.75, 1 minimum alveolar concentration (MAC))]. The peak of I-NMDA was recorded by means of the whole cell patch clamp technique. The cytochrome C level was detected by Western blotting and quantitative real-time PCR. Our results showed that isoflurane (0.25, 0.5, 0.75 and 1 MAC) potentiated the amplitude of I-NMDA by (116 +/- 8.8)%, (122 +/- 11.7)%, (135 +/- 14.3)% and (132 +/- 14.6)%, respectively, and isoflurane increased the mRNA expression of cytochrome C in a concentration-dependent manner. The cytochrome C mRNA expression reached a maximum after 0.5 MAC isoflurane stimulation for 6 h (P < 0.05). It was concluded that isoflurane enhances the expression of cytochrome C in cultured rat hippocampal neurons, which may be mediated by facilitation of NMDA receptor.