BACKGROUND: We intended to evaluate the diagnostic usefulness of a multiplex reverse transcriptase- PCR (mRT-PCR) assay kit under dual priming oligonucleotide system (DPO) for the childhood acute respiratory tract infections. METHODS: Two hundred nasopharyngeal aspirates were taken from children < or =5 yr old admitted due to acute respiratory infections in 2004. Direct fluorescent antibody (FA) assays were performed with fresh specimens; then, mRT-PCRs for the detection of 12 respiratory viruses (Seeplex RV detection kit, SeeGene, Seoul, Korea) were tested with frozen specimens. RESULTS: FA assays for five common respiratory viruses showed positive results in 66 patients (33.0%), while mRT-PCR detected causative viruses in 112 patients (56.0%), including 16 co-infected cases (8.0%). A total of 129 viruses were identified: respiratory syncytial virus A/B (38.0%/7.8%), influenza virus A/B (10.1%/5.4%), parainfluenza virus 1/2/3 (7.0%/3.1%/7.8%), coronavirus 229E or NL63 (6.2%), human metapneumovirus (4.7%), adenovirus (4.7%), rhinovirus (3.9%), and coronavirus OC43 (1.6%). CONCLUSIONS: DPO-based mRT-PCR was found as a sensitive tool for the detection of the viruses that cause childhood respiratory infections. Clinical significances of the agents detected by mRTPCR need further evaluations.