Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.