Two conserved regions (CR1 and CR2) on the adenovirus E1A proteins have previously been shown to be required for cooperation with the ras oncogene in the transformation of primary rodent cells. Sequences within these regions are essential for the ability of E1A to associate with the 105K product of the retinoblastoma susceptibility gene, p105-RB, as well as with other cellular proteins, including a 107K (p107) and a 300K (p300) species. In this paper, we show that CR1 mutants deficient in p300 binding and CR2 mutants with lost or reduced binding of p105-RB and/or p107 have a low, but not abolished focus formation activity. In contrast, CR1/CR2 double mutants were deficient in focus formation, suggesting that the transformation activities displayed by the single CR1 or CR2 mutants were due to an independent transformation activity by both CR1 and CR2. No strict correlation between p105-RB binding and E1A-mediated transformation was observed. The E1A enhancer repression function was found to correlate with the binding of p300 but not with E1A-mediated transformation. Complex formation between E1A and p107, similar to the p105-RB binding, required sequences within both CR1 and CR2. The CR2 sequences required for binding of p107K or p105-RB were overlapping, but not identical. Finally, a larger segment of CR2 was required for stable complex formation between E1A and phosphorylated forms of p105-RB or p107 compared to corresponding unphosphorylated species.