The creatine kinase-B (CKB) enzyme is proposed to have a pivotal role in the regeneration of ATP in the nervous system. In the present study, the steady-state levels of CKB mRNA were determined by RNase protection assay in seventeen separate brain regions obtained from rats during the initial interval of the light period or period of inactivity in rats. The antisense probe used specifically hybridizes to CKB mRNA and discriminates CKB from CKM mRNA. The results show that brain regions from Wistar rats differ in CKB mRNA content. Highest levels of CKB mRNA were detected in the male and female cerebellum. High levels of CKB mRNA were observed in the spinal cord, brain stem and its structures (medulla, pons and midbrain) and olfactory bulb of the male rats. Female rats also contained high levels of CKB mRNA in the brain stem. In both male and female rats, the frontal cortex, occipital cortex, hippocampus and striatum exhibited lower levels of CKB mRNA relative to the complete brain. Statistical analyses demonstrated a significant difference between the male and female CKB mRNA profiles. However, CKB mRNA levels in brain regions with estrogen receptors (hypothalamus, hippocampus) were similar in male and female rats. Differential CKB mRNA levels in various brain regions may suggest diverse physiological significance of the CKB system in the regulation of brain energy metabolism.