There is little information in the literature on the relationship among the frequency of carbohydrate consumption, the use of fluoride toothpaste, and enamel demineralization. The aim of this investigation was to compare the extent of demineralization of enamel slabs in situ, with a sugar-based solution, consumed in constant amounts but with various frequencies in subjects both with and without the use of fluoride (F) toothpaste. Eight subjects wore removable mandibular appliances carrying an enamel slab cut from white-spot lesions. The subjects were required to drink 500 mL of a 120-gm/L sugar solution either once, 3, 5, 7, or 10 times/day for 30 sec on each occasion, for a period of 5 days while brushing their teeth twice daily with either a F (1450 ppm NaF) or a F-free toothpaste. Mineral analysis revealed that when the subjects used a F toothpaste, net demineralization was evident only with the seven- and 10-times/day regime (ns). When F-free toothpaste was used, statistically significant demineralization was observed when the frequency exceeded 3 times/day. This study demonstrates the importance of F-containing toothpaste in enamel re-/demineralization by varying the frequency of carbohydrate challenge