Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.