Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood. Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl. We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C. M., and Vallano, M. L. (2002) J. Neurochem. 81, 314-324). CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.