We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 microM glutamate/10 microM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca(2+), respectively, to measure changes in [Ca(2+)](i). Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca(2+)](i) changes with exposure to 3 or 100 microM glutamate for 5 min, whereas magfura-2 reported larger [Ca(2+)](i) increases with 5-min exposure to 100 microM glutamate than with exposure to 3 microM glutamate, 100 microM kainate or 50 mM K(+) from 10 DIV onward. This suggests that the magnitude of the [Ca(2+)](i) changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca(2+). We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca(2+)](i) responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.