The in vitro life span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation of p53 was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of the p53 gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of the p53 gene during immortalization. The primary cells and the immortal derivatives all expressed authentic p53 mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5-8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes in p53.