Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18-mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA-positive and GPDH-rhodamine-positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture.