Background: In adults, measurement of F E NO has been recently suggested as a substitute for the methacholine challenge test (MCT) for diagnosis of asthma. This study aimed to evaluate whether FeNO is a substitute for MCH also in children with suspicious asthma. Methods: During a single visit steroid naive children (5-17 years) with suspicious asthma underwent skin prick test (SPT), F E NO measurement and spirometry prior and during MCT (one concentration procedure). Results of the SPT (atopy/non-atopy) and MCT (asthma/non-asthma) were used for categorization. ROC analysis in atopy non-atopy subgroups yielded sensitivity, specificity, positive and negative predictive value (PPV and NPV) for F E NO. Results: The SPT revealed atopy in 134 out of 222 children (age 9.7 ± 3.2 years) investigated and asthma was diagnosed in 114 (77/37 atopy/non-atopy) patients. F E NO values in patients with atopic asthma were significantly higher compared to those with either non-atopic asthma or atopia without asthma (18 ppb (5-89) vs 7 ppb (5-36); p < 0.001; 18 ppb (5-89) vs 11 ppb (5-98); p < 0.05). Sensitivity and specificity of F E NO for diagnosing atopic asthma (F E NO≥15.5 ppb; AUC = 0.635, p < 0.01) were 61.1% and 64.9% and non-atopic asthma (F E NO≥ 6.5 ppb; AUC = 0.445, p = 0.382) 54.1% and 39.2%, respectively. The PPV/NPV for F E NO were 0.70/0.55 in atopy and 0.39/0.54 in non-atopy patients, respectively. Conclusion: In children, F E NO is not appropriate to substitute for the MCT. However, in patients with a negative SPT a F E NO in the normal range makes the presence of atopic asthma unlikely.