BACKGROUND: Respiratory viruses are the main cause of acute respiratory tract infection (ARI) in children. Real-time polymerase chain reaction (PCR) technology is highly practicable for the rapid detection of viral pathogens. The simultaneous detection of a broad spectrum of viruses enables the diagnosis and evaluation of viral coinfection in ARI. METHODS: A 1-step real-time PCR was developed for the detection of 12 respiratory viruses (10 RNA and 2 DNA viruses) in clinical samples. Clinical samples from 254 children admitted to the Departments of Pediatrics with ARI during a 10-month period were tested. RESULTS: Respiratory syncytial virus (RSV) was the most frequently detected pathogen in 112 samples (44.1%), followed by human bocavirus (hBoV) in 49 (19.3%), and rhinovirus in 17 samples (6.7%). Viral coinfection was detected in 41 (16.1%) samples with RSV and hBoV being the most dominating combination (27 cases, 10.6%). Viral coinfection was found in 10 cases (17%) of children with bronchitis (n = 58) and in 7 cases (23%) of bronchiolitis (n = 30). In patients with pneumonia (n = 51), 17 cases (33%) were positive for 2 or more viral pathogens. CONCLUSIONS: Simultaneous testing of respiratory viruses by real-time PCR is a suitable tool for the detection of viral coinfections. In children hospitalized because of respiratory infection viral coinfection is frequently detected with RSV and hBoV being a common combination.