The cellular and intracellular localization of the epsilon-subspecies of protein kinase C (PKC) in the rat brain was demonstrated by immunocytochemistry using specific antibodies against epsilon-PKC. The epsilon-PKC-specific immunoreactivity was most abundant in the hippocampal formation, olfactory tubercle and Calleja's islands, was moderate in the cerebral cortex, anterior olfactory nuclei, accumbens nucleus, lateral septal nuclei and caudate-putamen and low in the thalamus and medulla. The epsilon-PKC-immunoreactivity was scanty in the perikarya, except for the pyramidal cells of CA3 region of the hippocampus and the immunoreactivity was mainly present in neuropils and nerve fibers. The distribution of epsilon-PKC immunoreactive neurons was consistent with that obtained by in situ hybridization histochemistry. Electron microscopic observations of the hippocampus revealed that the epsilon-PKC is predominantly present in the cytoplasm of axon and nerve terminals and that this enzyme is associated with mitochondrial membrane and vesicles. These results suggested that epsilon-PKC is probably involved in presynaptic functions in CNS, perhaps even neurotransmitter release.