The nucleotide sequences of the genomes of two strains of canine adenovirus, type 1 (Glaxo and CLL) have been determined within the region spanning approximately 0.80-0.91 map unit. Homology comparisons with other adenoviral DNAs indicate that this region contains part of the hexon-associated protein (pVIII) precursor gene on the left, and a 57-kDa fiber-encoding gene on the right. In both viral strains, the sequences of these genes are identical. Accepting the generally highly conserved nature of the genetic organization of adenoviral genomes, the 1136-bp sequence between them would include the canine equivalent of the human adenoviral E3 region. In the Glaxo strain, which is nonattenuated, this sequence contains only two R-strand open reading frames capable of coding for polypeptides of 60 residues or more. One of these appears to code for a transmembrane glycoprotein of 194 amino acid residues. Neither ORF, however, shows significant homology with any other known adenoviral DNA or protein sequence. The corresponding region in the attenuated strain CLL is almost identical with that of the Glaxo, but with a 325-bp deletion resulting in a fusion of the two ORFs. It seems, therefore, that neither ORF is essential for viral replication in vitro, since the CLL strain replicates just as efficiently as the Glaxo. The suggestion is made that the attenuation of the CLL strain may be related to the nonfunctionality of its E3 gene product(s).