The two major proteins of 243 amino acids (243R) and 289R encoded by the E1a region of adenovirus 5 are related, differing only by the presence of an internal conserved 46 amino acid segment in the 289R protein. We report here that these proteins have different cell transformation properties. Primary baby rat kidney (BRK) cells infected with an M-MuLV retrovirus vector that expresses the 243R protein are immortalized and the resulting foci consist predominantly of epithelial cells; a small fraction of the foci consist of fibroblast-like cells. In contrast, BRK cells infected with a retrovirus expressing the 289R protein are immortalized at a very low frequency and the foci induced consist of only fibroblast-like cells. The immortalizing properties of the 289R protein are dramatically changed by certain mutations within the unique 46 amino acid region: M-MuLV vectors with these mutations immortalize epithelial cells with high efficiency, as much as an order of magnitude higher than vectors that express the 243R protein. The enhanced immortalization property of the mutant forms of the 289R protein does not appear to be related to their transcriptional activation function. Our results suggest that the unique region of the 289R protein has the potential to inhibit immortalization of primary epithelial cells.