Infection with human parechovirus 3 (HPeV3) was described for the first time in Japan in 2004 and reportedly is more often associated with severe disease than infection with HPeV1 or HPeV2. In 2004, infections with HPeV3 were observed for the first time in The Netherlands. Genetic analysis showed several different lineages, suggesting endemic circulation. We analyzed 163 cell culture isolates from the same number of patients tested in routine virological laboratories as part of the national enterovirus surveillance program. Isolates were collected between 2000 and 2007 and could not be characterized by routine methods. In total, 155 isolates (95%) were found positive for HPeV by a reverse transcription-PCR assay targeting the 5 untranslated region, explaining the majority of the diagnostic deficit in enterovirus surveillance for these years. Typing of the isolates by use of partial genome sequencing of the VP1/2A region revealed the presence of 55 HPeV1, 2 HPeV2, 89 HPeV3, 1 HPeV4, and 8 HPeV5 isolates. We compared isolation dates, age groups affected, and clinical pictures, which were reported as part of the routine surveillance. Clear differences in epidemiology were observed, with HPeV3 occurring at intervals of 2 years and in the spring-summer season, whereas HPeV1 was observed in small numbers throughout each year, with a low in the summer months. HPeV3 infection affected younger children than HPeV1 infection and was significantly more often associated with fever, meningitis, and viremia.Human parechovirus 1 (HPeV1) and HPeV2, initially known as echovirus 22 (EV22) and EV23, were first isolated in the summer of 1956 in Japan from stool specimens of children suffering from diarrhea (9). From that time on, HPeV infections have been detected all over the world. Common symptoms caused by these viruses are gastroenteritis, respiratory infections, and, in a smaller proportion of cases (usually in children Ͻ1 year old), encephalitis and flaccid paralysis (12,20). Based on these disease symptoms and viral physicochemical properties (stability at a pH of Ͻ3 and in organic solvents), EV22 and -23 were initially grouped within the genus Enterovirus of the family Picornaviridae (18,19). Subsequent molecular characterization showed that the viruses were quite distant from the enteroviruses: EV22 and EV23 showed no more than 30% amino acid identity with other picornaviruses, and in contrast to most other human viruses in this family, they do not shut down the host cell protein translation machinery (9, 18). For these reasons, EV22 and -23 were grouped into a new genus of the Picornaviridae, Parechovirus, and were renamed HPeV1 and HPeV2. With this new genus, nine picornavirus genera are known at present: