Creatine kinase (CK) isoenzymes, with emphasis on the mitochondrial CK isoenzymes, were characterized and localized in chicken cerebellum. Chicken cerebellum extracts analyzed by two-dimensional gels, using antipeptide antibodies specific for sarcomeric muscle-type mitochondrial CK (Mib-CK) and revealed the presence of a Mib-CK variant in avian cerebellum. This CK isoform was localized by immunofluorescence staining exclusively in the Purkinje neurons. The co-expression of this Mib-CK together with cytosolic muscle-type MM-CK, as observed in the same Purkinje neurons, may reflect the specific energy requirements associated with highly fluctuating Ca2+ levels (Ca2+ spiking) in these specialized neurons. Ubiquitous brain-type mitochondrial Mia-CK was found together with cytosolic BB-CK mainly in the glomeruli structures of the cerebellar granular layer. BB-CK, but much less so Mia-CK however, was also very prominent in Bergmann glial cells of the two mitochondrial Mi-CK isoenzymes in the chicken cerebellum is demonstrated. Other hot spots of CK localization were the granule and pyramidal cells of the hippocampus in rat. There, a developmental stage-dependent immunofluorescence staining, especially with antibodies against Mia-CK was noted. Epithelial cells of the choroid plexus were also highly enriched in CK. The possible implications of a CK/PCr circuit at these various cellular locations of the brain are discussed with respect to normal brain physiology and pathology.