An adenovirus type 12 mutant, in203S, was constructed to contain an insertion of two amino acids in the early region 1A (E1A) 13S mRNA-coding region and in the E1A 12S mRNA intron. in203S could not grow in HeLa and KB cells. Virus DNA replication was scarcely detected at a low multiplicity of infection, but was detected at a high multiplicity of infection. The transcription of early genes other than E1A was not detected at 13 h after infection, but became detectable after longer incubation. The transcription of the E1A gene was also reduced to about one-fifth of the wild-type level. The mutant induced fewer foci of smaller sizes than the wild type in rat 3Y1 and secondary rat kidney cells. The induction of cellular DNA synthesis was reduced in rat 3Y1 cells infected with in203S as compared with that in wild type-infected cells. These results show that the E1A 13S mRNA-derived polypeptide of adenovirus type 12 is required for activation of early genes, cell transformation, and induction of cellular DNA synthesis.