It is well established that fluoride (F) prevents caries development by inhibiting demineralization and enhancing remineralization processes. However, it is not known which of these protective mechanisms is more important. In this double-blind, crossover in situ study conducted in three phases of 14 days each, 12 volunteers wore palatal appliances containing enamel and root dentin slabs, on which biofilm was allowed to accumulate under exposure to 20% sucrose solution 8×/day. F toothpaste was used once a day, either before the daily demineralizing episodes (in the morning) or after them (at night). Non-F placebo toothpaste was used in the control group. F toothpaste significantly reduced enamel and dentin demineralization compared with the control (p < 0.05). F toothpaste was more effective when used after the demineralization episodes than before, and this difference was statistically significant for dentin (p < 0.05). The results suggest that brushing with F dentifrice at night to remineralize daily mineral losses may be preferable to brushing in the morning to inhibit the demineralizing episodes of the day.