The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined. When compared to Ad5, a group C adenovirus, these sequences have been found to be 80% homologous. Most changes are transitions or transversions. This high degree of nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded from these genes; most nucleotide changes occur at the third position in the codon. The predicted polypeptide contains 448 amino acids and has a calculated Mr-value of 50700. The positions of the 5' end of the mRNA and of the donor and acceptor splice sites of Ad7 and Ad12 can be inferred by analogy to those of Ad5. A long open reading frame starting upstream from the IVa2 gene overlaps the N-terminal portion of the polypeptide but is encoded in a different reading frame. Within this overlapping region, the long open reading frame is more conserved in amino acid sequence than is the presumed IVa2 polypeptide, suggesting that evolutionary pressure was exerted on the longer protein, a product of viral early region 2B. The high degree of conservation of this E2B region within the overlapping segment suggests that its activities must be more important for adenovirus infection than are the functions encoded in the amino-terminus of the IVa2 gene.