In addition to the viruses belonging to the nine proposed genera of the Picornaviridae, Enterovirus, Rhinovirus, Cardiovirus, Aphtovirus, Hepatovirus, Parechovirus, Kobuvirus, Erbovirus and Teschovirus, two new members of this family have recently been discovered. Three strains of Ljungan virus (LV) were isolated from bank voles (Clethrionomys glareolus) and A-2 plaque virus (A-2) was isolated from human sera. To study the genetic relationship between these recently discovered viruses and the members of the family Picornaviridae, an evolutionary analysis has been carried out using the amino acid sequences of the two nonstructural proteins 2C and 3D. Phylogenetic analysis using prime members of the nine genera support the division of picornaviruses into the proposed genera. The study also supports a previous suggestion based on analysis of partial sequences of the structural proteins that LV is more related to the genus of Parechovirus than to other picornaviruses, but also shows that the three LV strains used in the comparison constitute a distinct monophyletic group, clearly separated from the parechoviruses. The analyses using the 2C and 3D sequences clearly showed that A-2 was related to the genera of Rhinovirus and Enterovirus, but it was not possible to group the A-2 with high confidence into one of the genera. Comparison using the VP1 protein sequences of Enterovirus and Rhinovirus showed that although the A-2 virus is positioned between the two genera, the virus is more related to the genus of Enterovirus than to Rhinovirus. Our analysis of the three LV strains based on the phylogenetic analysis of the 2C and 3D proteins suggests that the strains used in this study constitute a monophyletic group clearly related to Parechovirus of Picornaviridae. The taxonomic position of the A-2 virus is presently uncertain but available data indicate that this virus may be classified as a member of the genus of Enterovirus.