Since the effect of the combination of methods of fluoride use on enamel demineralization and on plaque composition is not clearly established, this study examined the effect of the combination of acidulated phosphate fluoride (APF) application and F dentifrice on enamel demineralization and on plaque composition. In this crossover study, 16 volunteers, wearing a palatal appliance containing bovine enamel blocks, were subjected to 4 treatment groups: non-fluoridated dentifrice (PD), FD, APF+PD, and APF+FD. The APF was applied to the enamel before the 14-day experimental period. During the experimental period, test dentifrices were applied 3x/day, and a 20% sucrose solution was applied 4x and 8x/day by being dripped on the blocks. Although APF application was able either to increase F concentration in plaque or to reduce the % of mutans streptococci, its combination with F dentifrice use neither reduced enamel mineral loss nor changed any other measured plaque variable with respect to the FD group alone.