Expression of the Ad5 E1A first exon is necessary and sufficient to cooperate with an activated RAS oncogene to transform primary epithelial cells. The second exon, although necessary for immortalization and induction of an epithelial cell growth factor, is not essential for co-transformation with T24 RAS. To determine whether the second exon has a role in the cooperation of E1A with an activated RAS gene, we have performed an extensive mutational analysis of this region. All of the deletion and point mutants that we have generated and analyzed retained the ability to enable the transformants to grow in serum-free media and in soft agar. A region in the C-terminus of the E1A polypeptide encoded by nucleotides 1437-1488 appears to modulate the level of transformation. Co-transfections of T24 RAS and E1A genes with mutations that bring about specific amino acid substitutions or deletions in the C-terminus result in enhanced transformation. There is an increase in the number of transformed foci and they appear earlier. A single amino acid change can bring about this phenotype, which is dominant over wild type. Thus, it seems that expression of the wild-type second exon retards or suppresses transformation. The hypertransforming phenotype does not correlate with any differences in the expression of the mutated E1A or the co-cotransfected RAS gene. The C-terminus encodes a nuclear localization signal for E1A, however the subcellular localization of the mutant polypeptides does not affect their co-transforming ability.