To determine the requirements for the individual Ad2 E1B proteins during the transformation of rodent cells, viral mutants were constructed with genetic lesions disrupting the coding sequence of either the 175 amino acid residue (175R) or the 495 amino acid residue (495R) E1B proteins. Point mutations generating stop codons very early in the coding sequences were constructed to prevent the expression of amino-terminal protein fragments which might have biological activity. Mutant virus pm1722 contains a point mutation that terminates translation of the 175R protein after three amino acids. It was completely defective for transformation of CREF cells in virion- and DNA-mediated assays. In HeLa cells, pm1722 replicated as well as wild-type virus but produced an extreme cytopathic effect and fragmentation of host-cell DNA. Nonetheless, we provide evidence that the observed transformation defect is not due to the death of transformed cells. The mutant virus dl1520, a double mutant unable to synthesize the 495R protein, was also extremely defective for the transformation of CREF cells in virion- and viral DNA-mediated assays. This result is in contrast to studies with other Ad5 mutants with lesions in the equivalent protein. Possible explanations for this difference are discussed. Replication of dl1520 in HeLa cells was significantly reduced compared to wild-type. Studies with a third mutant virus, pm2022, which contains a stop codon after the second codon of the 495R protein, suggest that very low levels of 495R protein activity are sufficient for a productive infection and significant transforming activity.