This in situ crossover and blind study was conducted to investigate the effect of professional acidulated phosphate fuoride (APF) gel application time on the subsequent inhibition of enamel demineralization. During 3 phases of 28 days each, 15 volunteers wore palatal appliances containing 4 enamel blocks, which were subjected to 3 treatment groups: not treated (control) and pre-treated with APF gel for 1 or 4 min. Dental plaque was allowed to accumulate on the blocks and the appliances were immersed in 10% sucrose solution 3 times a day simulating a cariogenic challenge. After each phase, the blocks were removed to evaluate enamel demineralization and concentration of fuoride (F) remaining after the cariogenic challenge. F formed on enamel was determined in additional enamel blocks subjected only to APF gel application. APF gel was effcient in reducing enamel demineralization (p<0.05), irrespective of the application time (p>0.05). Also, the concentration of the F formed and retained on enamel was signifcantly higher after APF gel application (p<0.05), but the effect of time of application was not statistically signifcant (p>0.05). The results suggest that APF application for either 1 or 4 min is equally effcient to increase F concentration in enamel and reduce enamel demineralization.