Rodent cells transformed by adenovirus 2 (Ad2) express two highly related viral proteins of 289 and 243 amino acids encoded in early region 1A (E1A). Transformation studies were performed with adenovirus mutants that express only one or the other E1A protein. We found that the 289 amino acid protein, which has transcription inducing activity, and the 243 amino acid protein, which has little if any of this activity, were both required to produce the fully transformed phenotype. Expression of either E1A protein induced a partially transformed phenotype. The 243 amino acid protein was particularly important for anchorage independent growth. As found in previous studies with several other E1A mutants, the process of transformation by the mutant that expresses the 243 amino acid protein only was cold-sensitive. While the 289 amino acid protein is the only E1A protein required for efficient viral replication under standard cell culture conditions, the 243 amino acid protein in addition to the 289 amino acid protein was found to be required for efficient viral replication in growth-arrested human cells.