During the early period of infection, adenovirus type 12 E1A gene is expressed as overlapping, spliced mRNAs of 12 and 13S, which encode in-frame proteins of 235 and 266 amino acid residues (235R and 266R), respectively. To define the functions of these related products in the infection of human cells and transformation of rodent cells, we created single T-to-C transitions at the second base of each mRNA intron which specifically prevent splicing of the respective mRNAs. Mutant pm712 expresses only the 13S mRNA and 266R protein, while pm713 expresses only the 12S mRNA and 235R protein. By using these mutants, we showed that only the larger product is required for growth in human cells, including growth-arrested W138 cells, that the capacity to activate other viral genes (in human cells, at least) lies primarily with that protein, and that the 266R product is not required for autoregulation of its own transcription. In the presence of the 266R protein the 235R product was not required for complete and efficient transformation of a variety of rodent cells or for direct induction of tumors in rats, whereas in its absence the smaller product was insufficient for transformation or tumor induction. Finally, we showed that transformants resulting from infection of rodent cells with pm712 possess a fully-transformed phenotype and are tumorigenic. Previous studies with group C adenoviruses led to the conclusion that both E1A products are required for complete transformation; we conclude that with oncogenic serotype 12, only the 266R product is required for this process.