RATIONALE: The diffusing capacity (DL) of the lung can be divided into two components: the diffusing capacity of the alveolar membrane (Dm) and the pulmonary capillary volume (Vc). DL is traditionally measured using a single-breath method, involving inhalation of carbon monoxide, and a breath hold of 8-10 seconds (DL,CO). This method does not easily allow calculation of Dm and Vc. An alternative single-breath method (DL,CO,NO), involving simultaneous inhalation of carbon monoxide and nitric oxide, and traditionally a shorter breath hold, allows calculation of Dm and Vc and the DL,NO/DL,CO ratio in a single respiratory maneuver. The clinical utility of Dm, Vc, and DL,NO/DL,CO in the pediatric age range is currently unknown but also restricted by lack of reference values. OBJECTIVES: The aim of this study was to establish reference ranges for the outcomes of DL,CO,NO with a 5 second breath hold, including the calculated outcomes Dm, Vc, and the DL,NO/DL,CO ratio, as well as to establish reference values for the outcomes of the traditional DL,CO method, with a 10 second breath hold in children. METHODS: DL,CO,NO and DL,CO were measured in healthy children, of European descent, aged 5-17 years using a Jaeger Masterscreen PFT. The data were analyzed using the Generalized Additive Models for Location Scale and Shape (GAMLSS) statistical method. MEASUREMENTS AND MAIN RESULTS: A total of 326 children were eligible for diffusing capacity measurements, resulting in 312 measurements of DL,CO,NO and 297 of DL,CO, respectively. Reference equations were established for the outcomes of DL,CO,NO and DL,CO, including the calculated values: Vc, Dm, and the DL,NO/DL,CO ratio. CONCLUSION: These reference values are based on the largest sample of children to date and may provide a basis for future studies of their clinical utility in differentiating between alterations in the pulmonary circulation and changes in the alveolar membrane in pediatric patients.