Restriction endonuclease cleavage maps have been constructed for the genome of a canine adenovirus type 1 (CAV-1) vaccine strain (CLL; Connaught Laboratories, Ltd., Willowdale, Ontario). Restriction enzyme analyses were also carried out on CAV-1 (CLL) genomes isolated from viral stocks over 8 serial passages in a dog kidney cell line (DK 6722). The right hand 20% of the genome became more heterogeneous in size with increasing passage in DK 6722 cells due to deletions up to 3-4 kb, whereas the left terminal region was stable throughout these passages. A comparative study of CAV-1(CLL) and a virulent strain of CAV-1, Glaxo, revealed that the genome of CAV-1(CLL) was the shorter, by about 480 bp, within the region covering 0.83-0.91 map units. By virtue of its location within the genome and its dispensable nature for viral growth, this region would appear to encompass a genetic sequence corresponding to the E3 region of human adenoviruses. In terms of viral attenuation, the possible importance of the observed differences between CAV-1(CLL) and CAV-1(Glaxo) is discussed.