GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins. In addition, we have recently demonstrated that calpain activation produces a truncation of GSK-3 that removes the N-terminal inhibitory domain (Goñi-Oliver et al. [2007] J. Biol. Chem. 282:22406). Given that calpain is involved in post-mortem proteolysis in brain samples, the objective of this investigation was to test whether GSK-3 is truncated in post-mortem samples. To achieve this objective, we first investigated the degradation of GSK-3 during different post-mortem intervals in mouse brains and found that the conversion of GSK-3 to proteolytic fragments of 40 and 30 kDa takes place in a way similar that of to p35-CDK-5 subunit and spectrin, two well-known calpain substrates. In addition, we demonstrated that this truncation is mediated by calpain, insofar as pretreatment with MDL 28170, a permeable blood-brain barrier calpain inhibitor, partially inhibited that degradation. When human brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of approximately 40 and 30 kDa, a proteolytic process that was inhibited by calpeptin, a specific calpain inhibitor. Thus, this is the first report of calcium-dependent truncation of human GSK-3. These data demonstrate that control samples with similar post-mortem delay are essential to interpret correctly the changes observed in GSK-3 levels in human post-mortem brain, especially when studying human neurodegenerative diseases.