Our previous work has shown that chronic ethanol treatment upregulated NMDA receptor function and binding in mammalian cortical neurons. However, the potential molecular mechanisms involved in these phenomenon have yet to be elucidated. In the present study, using RNase protection assay, we investigated the effect of chronic ethanol treatment on the NMDA receptor subunits R1, R2A, and R2B mRNA levels in cultured cortical neurons. We found that chronic ethanol (50 mM, 5 days) exposure did not change the NMDA receptor R1 and R2A subunits mRNA levels. In contrast, the NMDA receptor R2B subunit mRNA level was increased by approximately 40% with respect to the control values. The levels of the R2B subunit mRNA returned to the control values following the removal of ethanol for 72 h. In order to determine the involvement of the NMDA receptors in the action of chronic ethanol exposure, we further investigated the effect of the NMDA receptor antagonists on the upregulation induced by chronic ethanol exposure. The results indicate that the increased R2B subunit level was reversed by concomitant chronic exposure of the cortical neurons to the NMDA receptor competitive (10 microM; CPP), and non-competitive (1 microM; MK-801) antagonists, but not by the non-NMDA receptor antagonist, CNQX (10 microM), or the L-type calcium channel blocker, nitrendipine (10 microM). Taken together, these results suggested that chronic ethanol exposure selectively upregulated the NMDA receptor subunit R2B mRNA level in cortical neurons, and this increased NMDA receptor gene expression appears to be a NMDA receptor mediated process. The altered NMDA receptor gene expression may be responsible for the observed upregulation of the NMDA receptor binding and function in the cortical neurons following chronic ethanol exposure.