The early 3 (E3) transcriptional unit of human adenoviruses (HAV) encodes proteins that modulate host antiviral immune defenses. HAV E3 sequences are highly variable; different HAV groups encode phylogenetically unrelated proteins. The role of the E3 region of many human and animal adenoviruses is unknown because the sequences are unrelated to previously characterized viruses and the functions of proteins encoded by these regions have not been studied. We sequenced a portion of the bovine adenovirus serotype 1 (BAV-1) genome corresponding to the putative E3 region. This sequence was substantially different from other adenoviral E3 sequences, including those of two other bovine adenoviruses. However, two regions of putative sequence conservation were identified. BAV-1 E3 sequences were identified in early and late transcripts, but, unlike HAV, introns were not detected in the E3 region transcripts. Like HAV E3, a majority of the BAV-1 E3 region was not essential for growth in cell culture, as demonstrated by the construction of a recombinant BAV-1 lacking 60% of the putative E3 region.