Hyperphosphorylated tau protein is the primary component of neurofibrillary tangles observed in several neurodegenerative disorders. It has been hypothesized that in certain pathological conditions, the calcium activated protease, calpain, would cleave the cyclin-dependent kinase 5 (cdk5) activator p35 to a p25 fragment, which would lead to augmented cdk5 activity, and cdk5-mediated tau hyperphosphorylation. To test this hypothesis, we induced calpain-mediated p35 cleavage in rat hippocampal neuronal cultures and studied the relationship between p25 production, cdk5 activity, and tau phosphorylation. In glutamate-treated cells p35 was cleaved to p25 and this was associated with elevated cdk5 activity. However, tau phosphorylation was concomitantly decreased at multiple sites. The calpain inhibitor MDL28170 prevented the cleavage of p35 but had no effect on tau phosphorylation, suggesting that calpain-mediated processes, i.e., the cleavage of p35 to p25 and cdk5 activation, do not contribute to tau phosphorylation in these conditions. Treatment of the neuronal cultures with N-methyl-D-aspartic acid or with calcium ionophores resulted in an outcome highly similar to that of glutamate. We conclude that, in neuronal cells, the cleavage of p35 to p25 is associated with increased activity of cdk5 but not with tau hyperphosphorylation.