A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15-20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20-50 microM glutamate for 15 min was neurotoxic and induced a rapid (approximately 1-2 h) Ca(2+)-dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to > 800 microM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription-polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2- concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptors on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (< 20 microM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 microM calphostin C, 5 microM chelerythrine, or chronic exposure to 100 nM PMA. A 15-min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate-mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate-mediated neuronal death.