The mixed glial system of primary cultures of cells dissociated from neonatal rat brain was utilized to study glial differentiation. The investigation was addressed specifically to the possibility of noncoordinate regulation of two manifestations of oligodendroglial differentiation, i.e., activities of glycerol-3-phosphate dehydrogenase (GPDH) and of 2' ,3'-cyclic nucleotide 3'-phosphohydrolase (CNP), as well as the effects of initial cell density on the time of onset and the intensity of expression of these aspects of oligodendroglial differentiation. Simultaneously, glutamine synthetase activity was studied to determine effects on astrocytic differentiation. GPDH exhibited a major developmental increase in specific activity between 20 and 32 days in culture. However, CNP activity exhibited a major developmental increase that commenced approximately 2 weeks earlier. The onset of these expressions of oligodendroglial differentiation was not affected by such environmental factors as initial cell density. However, the intensity of expression of the temporally separate increases in GPDH and CNP activities was markedly density-dependent. The highest activities were attained in cultures plated at the lowest cell densities. The astrocytic enzyme, glutamine synthetase, also exhibited a striking developmental increase (approximately tenfold between 13 and 30 days in culture), but initial cell density affected neither the time of onset nor the intensity of expression of this aspect of astrocytic differentiation. The data demonstrate a striking developmental increase in GPDH activity that is not coordinate with that in CNP. The noncoordinate manifestations of oligodendroglial differentiation commence as a function of time in culture, whereas the intensity of expression of this differentiation can be influenced by such environmental factors as initial cell density. Neither the onset of nor the extent of the sharp increase in glutamine synthetase activity, an expression of astrocytic differentiation, is affected by initial cell density.