Immortalized central nervous system (CNS) cell lines are useful as in vitro models for innumerable purposes such as elucidating biochemical pathways, studies of effects of drugs, and ultimately, such cells may also be useful for neural transplantation. The SV40 large T (LT) oncoprotein, commonly used for immortalization, interacts with several cell cycle regulatory factors, including binding and inactivating p53 and retinoblastoma family cell-cycle regulators. In an attempt to define the minimal requirements of SV40 T antigen for immortalizing cells of CNS origin, we constructed T155c, encoding the N-terminal 155 amino acids of LT. The p53 binding region is known to reside in the C-terminal region of LT. An additional series of mutants was produced to further narrow the molecular targets for immortalization, and plasmid vectors were constructed for each. In a p53 temperature sensitive cell line model, T64-7B, expression of T155c and all constructs having mutations outside of the first 82 amino acids were capable of overriding cell-cycle block at the non-permissive growth temperature. Several cell lines were produced from fetal rat mesencephalic and cerebral cortical cultures using the T155c construct. The E107K construct contained a mutation in the Rb binding region, but was nonetheless capable of overcoming cell cycle block in T64-7B cell and immortalizing primary cultured cells. Cells immortalized with T155c were often highly dependent on the presence of bFGF for growth. Telomerase activity, telomere length, growth rates, and integrity of the p53 gene in cells immortalized with T155c did not change over 100 population doublings in culture, indicating that cells immortalized with T155c were generally stable during long periods of continuous culture.