Using primary cultures of murine cerebral cortices, we investigated the developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in relation to the appearance of NMDA receptor-mediated glutamate neurotoxicity. The cultures were not affected by glutamate exposure on culture days 7-9, but became sensitive to glutamate neurotoxicity on day 11. The expression of NMDA receptor subunit messenger RNAs (mRNAs) was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The epsilon 3-NR2C and epsilon 4-NR2D transcripts could not be detected in the culture. The epsilon 2-NR2B and zeta 1-NR1 subunit mRNAs, on the other hand, could be detected clearly and continuously from the culture initiation, and the epsilon 1-NR2A subunit mRNA became clearly detectable on culture day 4. The expression of these three subunits' proteins in the glutamate-insensitive stage (culture day 8) and the sensitive stage (day 11) were studied by means of Western blotting. The epsilon 2-NR2B and zeta 1-NR1 subunit proteins were clearly expressed on culture days 8 and 11, but the epsilon 1-NR2A subunit protein could hardly be detected on either day 8 or day 11. These results suggest that the glutamate neurotoxicity in the primary culture was mediated mainly by epsilon 2/zeta 1 NMDA receptors. The time lag between the protein expression of the epsilon 2-NR2B and zeta 1-NR1 subunits and the emergence of glutamate neurotoxicity may be necessary for the maturation of functional NMDA receptor systems, including heteromeric receptor formation, increase in receptor density and maturation of the postreceptor signal transduction system.