Cow's milk allergy (CMA) is the most common food allergy in early childhood. The golden standard for the diagnosis of CMA is a food challenge after a period of elimination. Increased levels of fractional exhaled nitric oxide (FE(NO)) have been shown after bronchial allergen provocation. We evaluated whether FE(NO) may also be a predictor of a positive reaction during cow's milk challenge in infants. Forty-four infants [mean age (range): 4.2 (3.7-4.6) months] suspected of CMA underwent an open food challenge with cow's milk formula administered in ascending quantities, starting with 2 ml and then 6, 20, 60 and 200 ml until a clinical reaction occurred. Off-line FE(NO) samples were obtained during tidal breathing by means of a facemask covering infants' nose and mouth. FE(NO) was measured twice before the challenge (baseline), immediately before each new dose of milk and after a positive reaction or after the last dose of milk. Eleven children showed immediate positive clinical responses to cow's milk, whereas 13 infants presented only a late-type reaction. FE(NO) values before or after a positive reaction (either immediate or late) were not different from FE(NO) values at baseline. Baseline FE(NO) in infants with a positive reaction did not differ from FE(NO) in infants without a reaction at any time point. We conclude that FE(NO) values are not predictive and not related to the occurrence of a positive reaction during a cow's milk challenge in infants, suggesting that a positive reaction may not result from eosinophilic activation.