Purpose: To assess urinary fluoride excretion after topical application of a commercial fluoride varnish in preschool children. Materials and Methods: Twelve healthy children, 2.5-6.0 years of age, were enrolled in a placebo-controlled cross-over trial. After a 5-day run-in period, the morning urine was collected as baseline. One hour after breakfast, 0.1 ml of the active (Fluor Protector S; 7,700 ppm F) or the placebo varnish was applied with a microbrush on the buccal surfaces of the primary teeth. Thereafter, a 6-h urine sample was collected and the fluoride content was determined with an ion-sensitive electrode. The parents supervised toothbrushing with a small-fingernail amount of fluoride toothpaste (1000 ppm) twice daily during the entire experiment. Results: One boy failed to comply with the urinary samplings and was excluded. The mean fluoride concentration in the 6-h urine samples was slightly higher after the active varnish compared with the placebo varnish, but the difference was not statistically significant. Likewise, no statistically significant differences were obtained when the post-treatment concentrations were compared with baseline for the two varnishes. No side-effects or adverse events were reported. Conclusion: A single topical treatment with the investigated varnish did not significantly increase the urinary fluoride excretion compared with placebo in preschool children with parallel use of fluoride toothpaste. Keywords: caries prevention, dental varnish, fluoride excretion, preschool children, urine