DNA segments containing the major promoter at coordinate 16.5 for rightward transcription from human adenovirus serotypes 3 and 7 (Ad3 and Ad7), two closely related class B viruses, have been sequenced and found virtually identical. Furthermore, over 80% of the nucleotides of Ad3 and Ad7 in this entire region are homologous to their counterparts in the DNA of the more distantly related class C serotype Ad2. There are the same number of nucleotide pairs among these serotypes within the region compared. Most changes are transitions or transversions and the several single-base deletions are always compensated by nearby insertions. These few changes nonetheless result in 24 differences between Ad7 (or Ad3) and Ad2 in a total of 32 cleavage sites. The promoter for the rightward-transcribed RNAs and the first segment of the consanguinous tripartite leader found at the 5'-ends of all the later mRNAs derived from that promoter have been identified by analogy to the nucleotide sequences of Ad2. In particular, the "Hogness box" or RNA polymerase staging site for the major rightward transcription unit is completely homologous to that of Ad2. There are only six bp changes in the first late leader segment despite previous evidence suggesting that they might be quite heterologous. A prominent dyad axis of symmetry exists just upstream from the presumed 5'-end of the late RNA. However, unlike the stem-loop structure proposed for Ad2 by Ziff and Evans (1978), the base changes relative to Ad2 mandate a different potential stemloop structure in the single strand of Ad3 and Ad7 DNAs. This hairpin places the "Hogness box" immediately next to the 5'-end of the RNA at the base of stem. An analogous dyad axis of symmetry or stemloop structure can be found in a number of eukaryotic systems, including the major rightward transcription unit of Ad2. This feature may be of relevance to the positioning of RNA polymerase II on the DNA and to the promotion of transcription.