Glutamate induces gene transcription in numerous physiological and pathological conditions. Among the glutamate-responsive transcription factors, NF-κB has been mainly implicated in neuronal survival and death. Recent data also suggest a role of NF-κB in neural development and memory formation. In non-neuronal cells, degradation of the inhibitor IκBα represents a key step in NF-κB activation. However, little is known of how glutamate activates NF-κB in neurons. To investigate the signalling cascade involved we used primary murine cerebellar granule cells. Glutamate induced a rapid reduction of IκBα levels and nuclear translocation of the NF-κB subunit p65. The glutamate-induced reduction of IκBα levels was blocked by the N-methyl-d-aspartate inhibitor MK801. Specific inhibitors of the proteasome, caspase 3, and the phosphoinositide 3-kinase had no effect on glutamate-induced IκBα degradation. However, inhibition of the glutamate-activated Ca2+-dependent protease calpain by calpeptin completely blocked IκBα degradation and reduced the nuclear translocation of p65. Calpeptin also partially blocked glutamate-induced cell death. Our data indicate that the Ca2+-dependent protease calpain is involved in the NF-κB activation in neurons in response to N-methyl-d-aspartate receptor occupancy by glutamate. NF-κB activation by calpain may mediate the long-term effects of glutamate on neuron survival or memory formation.