Four strains of canine adenovirus type 1 (CAV-1) and two strains of canine adenovirus type 2 (CAV-2) were examined by restriction enzyme analysis. In all cases, CAV-1 could be readily differentiated from CAV-2. Additionally, monoclonal antibodies were prepared against the Mirandola and Manhattan strains of CAV-1 and CAV-2, respectively. 18 of 36 monoclonal antibodies from the CAV-1 fusion and 77 of 160 monoclonal antibodies from the CAV-2 fusion were type-specific by an indirect fluorescent-antibody technique. Moreover, among those type-specific monoclonal antibodies, 13 of the 18 CAV-1 antibodies and 39 of the 77 CAV-2 antibodies specifically neutralized only the homologous virus in vitro. These data, along with other evidence from the literature, suggest that not only should CAV-1 and CAV-2 be recognized as distinct species in the genus mastadenovirus of the family Adenoviridae, but also that the major criterion of species distinction in that family, namely neutralization, should be reconsidered.