Cyclin-dependent kinase 5 (cdk5) regulates crucial neurobiological events, and deregulation of cdk5 has been implicated in several neurodegenerative disorders. The deregulation is suggested to occur due to cleavage of the cdk5 activator protein p35 to a smaller p25 fragment by the calcium-activated protease calpain. Here we have elucidated the role of different calcium-permeable ionotropic glutamate receptors in the induction of p35 cleavage in cultured rat hippocampal neurons. The glutamate receptor agonists glutamic acid, N-methyl-D-aspartate (NMDA), kainic acid, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were all able to induce p35 cleavage, in a manner depending on extracellular calcium. The effect of glutamate was mediated by NMDA receptors, as it was prevented by the NMDA antagonist MK-801. Cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization, enhanced glutamate-induced p35 cleavage. In immature 6-day-old cultures the non-NMDA agonist kainic acid provoked p35 cleavage, whereas glutamate and NMDA were ineffective. The data suggest that both NMDA and non-NMDA receptors are able to induce p35 cleavage. Different factors, such as maturation state of neurons or desensitization properties of non-NMDA receptors, may determine which receptor predominantly mediates the effect of glutamate on p35 cleavage.