Background: The relation between viral load and disease severity in childhood acute respiratory tract infections (ARI) is not fully understood. Objectives: To assess the clinical relevance of the relation between viral load, determined by cycle threshold (CT) value of real-time reverse transcription-polymerase chain reaction assays and disease severity in children with single-and multiple viral ARI. Study design: 582 children with ARI were prospectively followed and tested for 15 viruses. Correlations were calculated between CT values and clinical parameters. Results: In single viral ARI, statistically significant correlations were found between viral loads of Respiratory Syncytial Virus (RSV) and hospitalization and between viral loads of Human Coronavirus (HCoV) and a disease severity score. In multiple-viral ARI, statistically significant correlations between viral load and clinical parameters were found. In RSV-Rhinovirus (RV) multiple infections, a low viral load of RV was correlated with a high length of hospital stay and a high duration of extra oxygen use. The mean CT value for RV, HCoV and Parainfluenza virus was significantly lower in single-versus multiple infections. Conclusion: Although correlations between CT values and clinical parameters in patients with single and multiple viral infection were found, the clinical importance of these findings is limited because individual differences in host-,viral and laboratory factors complicate the interpretation of statistically significant findings. In multiple infections, viral load cannot be used to differentiate between disease causing virus and innocent bystanders. (C) 2017 Elsevier B.V. All rights reserved.