Analysis of canine parvovirus (CPV) isolates with a panel of monoclonal antibodies showed that after 1986, most viruses isolated from dogs in many parts of the United States differed antigenically from the viruses isolated prior to that date. The new antigenic type (designated CPV type 2b) has largely replaced the previous antigenic type (CPV type 2a) among virus isolates from the United States. This represents the second occurrence of a new antigenic type of this DNA virus since its emergence in 1978, as the original CPV type (CPV type 2) had previously been replaced between 1979 and 1981 by the CPV type 2a strain. DNA sequence comparisons showed that CPV types 2b and 2a differed by as few as two nonsynonymous (amino acid-changing) nucleotide substitutions in the VP-1 and VP-2 capsid protein genes. One mutation, resulting in an Asn-Asp difference at residue 426 in the VP-2 sequence, was shown by comparison with a neutralization-escape mutant selected with a non-CPV type 2b-reactive monoclonal antibody to determine the antigenic change. The mutation selected by that monoclonal antibody, a His-Tyr difference in VP-2 amino acid 222, was immediately adjacent to residue 426 in the three-dimensional structure of the CPV capsid. The CPV type 2b isolates are phylogenetically closely related to the CPV type 2a isolates and are probably derived from a common ancestor. Phylogenetic analysis showed a progressive evolution away from the original CPV type. This pattern of viral evolution appears most similar to that seen in some influenza A viruses.