Over‐activation of the N‐methyl‐d‐aspartate (NMDA) receptor results in a Ca2+‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca2+ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca2+ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced 45Ca2+ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and 45Ca2+ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to 45Ca2+ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca2+ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures.Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.