The aim of this study was to establish reference values and to examine day-to-day and within-day variations of exhaled nitric oxide (eNO) during tidal breathing in healthy children using a newly described method. Exhaled NO was measured on-line and off-line during tidal breathing through a facemask. In a subgroup of children measurements were repeated during the course of a single day and on the same time on three consecutive days. A total of 133 healthy children were included in the study and measurements were obtained from 121 children aged 2-7 yr (61 boys and 60 girls). The geometric mean eNO concentration and 95% CI was 3.9 (3.5-4.2) parts per billion (p.p.b.) for on-line measurements and 3.0 (2.7-3.3) p.p.b. for off-line measurements. Exhaled NO was independent of gender, age, height and weight. The 95% reference intervals (RI) for on-line and off-line measurements were 1.2-8.2 and 1.3-7.1 p.p.b. respectively. Twenty-three children completed measurements of within-day and day-to-day variations, none of which showed significant variation. In conclusion, the established reference values and data on variability within and between days may facilitate the clinical application for measurement of eNO during tidal breathing in young children.