Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.