Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.