BACKGROUND: Human coronavirus NL63 (HCoV-NL63) has recently been implicated as a common cause of croup in children. This study was performed to evaluate viruses associated with croup in children, with an emphasis on HCoV-NL63. METHODS: Nasopharyngeal aspirates were prospectively collected from 182 children hospitalized with croup at Seoul National University Bundang Hospital from January 2005 to June 2009. Multiplex reverse-transcriptase polymerase chain reaction was conducted for detection of 11 respiratory viruses, and medical records were reviewed. RESULTS: Viruses were identified in 147 (80.8%) of the 182 croup patients. The 3 most commonly detected viruses were parainfluenza virus type 1 (PIV1) in 44 (24.2%) patients, HCoV-NL63 in 30 (16.5%) patients, and influenza A virus in 25 (13.7%) patients. Other detected viruses were rhinovirus in 22 (12.1%) patients, PIV type 3 and respiratory syncytial virus in 15 (8.2%) patients, human bocavirus in 8 (4.4%) patients, and several others in a few patients. Coinfections with > or = 2 viruses were found in 20 (11%) patients. HCoV-NL63 was identified primarily in winter, which coincides with the peak occurrence of croup. Patients with HCoV-NL63 infection were younger than those who were positive for PIV1 (median age 13 months versus 21 months, P = 0.006) and had shorter fever duration than influenza A virus infection (median 1.5 days versus 4 days, P = 0.000). CONCLUSIONS: PIV1 and HCoV-NL63 were most commonly associated with patients who were hospitalized with croup. HCoV-NL63 is prevalent in winter and is associated with younger age and with shorter fever duration.