Measurement of the fraction of exhaled nitric oxide (FeNO) is a useful test to diagnose and/or monitor eosinophilic airway inflammation. The off-line tidal breathing method is used for measurements in young children, but reference values for preschool children are scarce. The objective of this study was to establish reference values for FeNO in healthy children 1-5 years old. We included 51 healthy children (23 males), mean age 32.5 months, from the general population and measured FeNO, using an off-line tidal breathing method with a chemiluminescence analyzer. The method proved feasible in 100% of the children. Geometric mean FeNO was 7.1 parts per billion (ppb), 95% confidence interval 2.8-11.5 ppb, with the 95th percentile 22.6 ppb. No significant difference was found between boys and girls, and no correlations were observed between FeNO and age, height, or weight. This study demonstrates that the off-line tidal breathing method is feasible to measure FeNO in preschool children and provides reference values of FeNO in healthy children 1-5 years of age.