Alternative splicing of beta-amyloid precursor protein (APP) RNA generates APP isoforms with or without a Kunitz protease inhibitor (KPI) domain. Previously, we showed that KPI (+) APP RNA, but not KPI (-) APP RNA, is upregulated in response to experimental lesions in which neurotoxicity is dependent on NMDA receptor activation and in Alzheimer's disease hippocampus. Recent studies by Mucke et al. (1995) showed that neuronal expression of human KPI (+) APP, but not KPI (-) APP, in transgenic mice is neuroprotective against experimental lesions. In this study we examined the direct effects of the excitotoxic amino acid Glu on alternatively, spliced APP RNAs and the corresponding protein isoforms in cultured rat cortical neurons. Glu treatment rapidly induced (4.5 h) KPI (+) APP RNA but not KPI (-) APP RNA. Induction of KPI (+) RNA preceded Glu-induced neuronal cell death and was partially blocked by an NMDA-receptor antagonist. In contrast to the RNA, cellular levels of KPI (+) APP were not changed by 4.5 h of Glu treatment. Instead, the cellular full-length form of the protein KPI (-) APP was reduced by approximately 50% after 2 h of Glu treatment and remained depleted after 24 h of treatment. Cellular levels of KPI (+) forms of amyloid precursor-like protein 2 (APLP2) were not changed by Glu treatment. Our data are consistent with the hypothesis that sustained NMDA-receptor activation can regulate alternative splicing of the APP pre-mRNA in neurons.