We examined properties of glia derived from very early neurogenesis in the chick embryo, as well as their behavior in response to extended cell passages and at various periods in culture. Primary cultures derived from the telencephalic region of 3-day-old chick embryo (stage 19) exhibited intense staining for vimentin (Vim; indicative of immature glial phenotypes) throughout the 17-day culture period, transitioning to Vim-positive/glial fibrillary acidic protein (GFAP)-positive astroblasts after a single cell passage at 13 days in vitro (DIV). With subsequent passage (P; through P6), cell continued to coexpress Vim/GFAP with the occasional appearance of fibronectin (Fib). By P11, Vim staining was faint, whereas GFAP staining gained in intensity, indicating the presence of mature astrocytes. These cultures also featured significant activity of glutamine synthetase (GS), which increased slightly with both cell passage and days in culture, correlating well with immunocytochemical findings. The activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP) remained low, indicating a low percentage of mature oligodendrocytes. Thus, embryonic day (E)3H glia cultures mature into astrocytes given sufficient time in culture. To obtain a stable population of glia at various stages of astrocytic differentiation, we immortalized and subcloned homogenous colonies of E3H glia precursors at specific stages of development. A cell suspension was electroporetically transfected with the pSV40neo gene for large T antigen (E3H.SV5). After 5 passages, the parent colonies consisted of a heterogeneous population of cells, most of which were vim and GFAP positive. Following subcloning, one line (colony 7) displayed the staining pattern of mature astrocytes. However, with advancing age in culture, staining for both vim and GFAP became increasingly faint. Compared with control (normal) cultures, transfected cells exhibited a significantly lower activity of GS that did not fluctuate with days in culture but decreased with advancing cell passage. Furthermore, CNP activity in E3H.SV5 colony 7 was approximately double that observed in normal cultures at the same cell passage. This observation suggests that the phenotype of transfected glia was less stable than that observed in normal glial cultures.