Duck hepatitis virus type 1 (DHV-1) was previously classified as an enterovirus, based primarily on observed morphology and physicochemical properties of the virion. The complete nucleotide sequences of two strains (DRL-62 and R85952) of DHV-1 have been determined. Excluding the poly(A) tail, the genomes are 7691 and 7690 nt, respectively, and contain a single, large open reading frame encoding a polyprotein of 2249 aa. The genome of DHV-1 is organized as are those of members of the family Picornaviridae: 5' untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR. Analysis of the genomic and predicted polyprotein sequences revealed several unusual features, including the absence of a predicted maturation cleavage of VP0, the presence of two unrelated 2A protein motifs and a 3' UTR extended markedly compared with that of any other picornavirus. The 2A1 protein motif is related to the 2A protein type of the genus Aphthovirus and the adjacent 2A2 protein is related to the 2A protein type present in the genus Parechovirus. Phylogenetic analysis using the 3D protein sequence shows that the two DHV-1 strains are related more closely to members of the genus Parechovirus than to other picornaviruses. However, the two DHV-1 strains form a monophyletic group, clearly distinct from members of the genus Parechovirus.