Adenovirus early region 1A (E1A) products induce DNA synthesis, transform primary rodent cells, and activate transcription factor E2F through complex formation with an array of cellular proteins via the E1A amino terminus and conserved regions 1 and 2 (CR1 and CR2). Interactions with the retinoblastoma tumor suppressor, pRb, and related proteins p107 and p130 rely somewhat on CR1 but largely on CR2, which contains a core binding sequence Leu-122-X-Cys-X-Glu. We introduced point mutations in CR2 to define such interactions more precisely. In human cells, alteration of any of the conserved residues within the binding core eliminated complex formation with pRb. Conversion of nonconserved Thr-123 to Pro (but not to either Ala or Ser) disrupted binding of pRb, presumably because of conformational changes in the binding core. No single E1A point mutant was completely defective in binding p107, suggesting that molecular interactions between E1A proteins and p107 clearly differ from those with pRb and p130. In general, the patterns of complex formation by E1A mutants in rat, monkey, and human cells were quite similar. All mutants which failed to bind significant amounts of pRb also failed to transform primary rat cells. Several mutants demonstrated selective binding to pRb, p107, and p130, but transforming activity corresponded largely with complex formation with pRb, regardless of the levels of interactions with p107 and p130. Mutants defective for binding of both pRb and p107 failed to induce the activity of transcription factor E2F; however, quite high levels were activated by E1A mutants that interacted with p107 alone. These results suggested that both pRb and p107 are important regulators of E2F activity but that complex formation with and activation of E2F by p107 are insufficient for cell transformation.