The physiological role of the phosphocreatine (PCr)/creatine kinase (CK) system has been studied in rat brain by comparing maturational changes in in vivo CK-catalyzed reaction rate and activities of CK isoenzymes. The CK-catalyzed reaction rates, measured by 31P-nuclear magnetic resonance spectroscopy, increased 4-fold between 12 and 17 days of age. The mitochondrial CK (Mi-CK) isoenzyme, as a percentage of total CK, increased to the same extent over this relatively narrow age period. Cytosolic CK (B-CK) was active earlier and, with the total CK activity, increased steadily over a longer time course. An immunohistochemical study of cerebellum showed Mi-CK predominantly in gray matter, while the cytosolic CK was present in rather large concentrations in both gray and white matter. In the molecular layer, B-CK was most prominent in the Bergmann glial cells, while Mi-CK was more prominent in Purkinje neurons. During development a redistribution of Mi-CK from the Purkinje cell bodies to their processes was observed. These results point to regional differences in CK content and in isoenzyme-specific localizations. The increase in CK activity is temporally coincident with the maturational appearance of closely coupled decreases in brain PCr and ATP during hypoxia. These maturational changes suggest that the activity of the PCr/CK system, particularly the Mi-CK isoenzyme, is central in regulation of brain ATP.