The early transcription region 3 (E3) of group B adenovirus type 35 (Ad35), a serotype isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders, has been partially sequenced. We had previously identified an Ad35 29-kilodalton (kDa) early glycoprotein which, analogous to group C Ad2 E3-19K, associated with major histocompatibility complex class I antigens in the endoplasmic reticulum of infected cells. The open reading frame (ORF) of the Ad35 29-kDa protein has now been identified within a 2-kilobase-pair cloned Ad35 E3 fragment. The predicted amino acid sequence was very similar to that of group B Ad3 E3-19K. In contrast, homology between the Ad35 and Ad2 glycoproteins was limited to five cysteines in identical positions and a 20-amino-acid region proximal to the transmembrane domain. In addition, 20.3- and 20.6-kDa ORFs have been identified downstream from the ORF for the Ad35 glycoprotein. Analogous 20-kDa ORFs are present in the Ad3 E3 region but are not present in Ad2 and Ad5. In contrast, the region analogous to an Ad2 11.6-kDa ORF, which is 9 kDa in size in Ad3, was absent from the expected position within the Ad35 E3 region. Because the E3 region is likely to play an important role in the interaction between virus and host, analysis of the function of the Ad35 E3 proteins should further our understanding of adenovirus pathogenesis.