As there is no homeostatic mechanism for maintaining circulating fluoride (F) in the human body, the concentration may decrease and increase again when intake is interrupted and re-started. The present study prospectively evaluated this process in children exposed to F intake from water and toothpaste, using F in urine as a biomarker. Eleven children from Ibiá, Brazil (with sub-optimally fluoridated water supply) aged two to four years who regularly used fluoridated toothpaste (1,100 ppm F) took part in the study. Twenty-four-hour urine was collected at baseline (Day 0, F exposure from water and toothpaste) as well as after the interruption of fluoride intake from water and dentifrice (Days 1 to 28) (F interruption) and after fluoride intake from these sources had been re-established (Days 29 to 34) (F re-exposure). Urinary volume was measured, fluoride concentration was determined and the amount of fluoride excreted was calculated and expressed in mg F/day. Urinary fluoride excretion (UFE) during the periods of fluoride exposure, interruption and re-exposure was analyzed using the Wilcoxon test. Mean UFE was 0.25 mg F/day (SD: 0.15) at baseline, dropped to a mean of 0.14 mg F/day during F interruption (SD: 0.07; range: 0.11 to 0.17 mg F/day) and rose to 0.21 (SD: 0.09) and 0.19 (SD: 0.08) following F re-exposure. The difference between baseline UFE and the period of F interruption was statistically significant (p < 0.05), while the difference between baseline and the period of F re-exposure was non-significant (p > 0.05). The findings suggest that circulating F in the body of young children rapidly decreases in the first 24 hours and again increases very fast after discontinuation and re-exposure of F from water and toothpaste.