The DNA of bovine adenovirus (BAV) serotype 4, a member of the subgroup 2 BAVs, has been cloned and mapped with 11 restriction enzymes. Southern blot hybridizations probed by a clone containing about 50% of the BAV-4 genome revealed a very strong and extended DNA sequence homology amongst the members of subgroup 2, but no homology was detectable to the subgroup 1 bovine, or any of those human (HAV) and porcine adenovirus serotypes examined. These findings were strengthened by reciprocal hybridizations. When using the cloned hexon gene region of BAV-3 (subgroup 1) or the total genome of HAV-2 as probes, again no homology could be shown to the bovine subgroup 2 serotypes. The extent of DNA homology detectable between the members of bovine subgroup 1, the porcine and the human serotypes was variable, but in general less expressed than that observed within the bovine subgroup 2.