Creatine kinase (CK) catalyzes a reversible transphosphorylation reaction that is believed to play a crucial role in the maintenance and channeling of high energy phosphate in tissues with high energy demands. In rat brain development, cytosolic (nonmitochondrial) CK levels increase rapidly during the peak period of myelination and remain high in the adult. The cellular compartmentation of CK was investigated through the use of primary cultures of neurons, oligodendrocytes and astrocytes. The CK activity in cultured oligodendrocytes, which expressed both enzymatic and immunocytochemical markers of myelin, was the highest of the cell types examined and comparable to levels measured in whole adult brain; these observations suggest a role for CK in myelinogenesis. We found that low density, dividing astrocyte cultures also exhibited high B-CK (brain isoenzyme of CK) immunoreactivity, with the nuclear CK staining being especially intense. We studied these cultured astrocytes in more detail with respect to their intense nuclear CK immunoreactivity. Optical sections of astrocyte nuclei taken with a confocal microscope show that the high B-CK present is actually contained within the nucleus, with a nucleoplasmic distribution that does not co-localize with DNA or RNA. To ascertain whether the high nuclear B-CK in proliferating astrocytes correlated with ongoing cell division, we conducted experiments with confluent, nondividing cultures. These results show that both CK enzyme activity and immunoreactivity are high in the nucleus of proliferating astrocytes, and significantly reduced in the nucleus of quiescent, nondividing astrocytes. The high level of CK protein and activity in the nucleus of proliferating astrocytes suggests a role for CK in cell division/nuclear function.(ABSTRACT TRUNCATED AT 250 WORDS)