Although the effect of acidulated phosphate fluoride gel (APF gel) on caries reduction in permanent teeth is based on evidence, the relevance of the clinical application time is still under debate. Also, the effect of 4- versus 1-min application has not been evaluated in deciduous enamel. In a blind, crossover, in situ study of 14 days, 16 adult volunteers wore palatal appliances containing slabs of human permanent and deciduous enamel. At the beginning of each phase, the slabs were submitted to one of the following treatments: no APF application (negative control); APF gel (1.23% F) application for 1 or 4 min. Biofilm accumulation on the slab surface was allowed and the slabs were subjected eight times a day to 20% sucrose, simulating a high cariogenic challenge condition. On the 15th day of each phase, fluoride retained as CaF(2) and fluorapatite (FAp) was determined on the enamel of the slabs and demineralization was assessed by cross-sectional microhardness. Fluoride as CaF(2) and FAp, formed by APF gel application on the enamel slabs not subjected to the cariogenic challenge, was also determined. APF gel reduced demineralization in both enamel types (p < 0.05), but the difference between 1 and 4 min was not statistically significant (p > 0.05). CaF(2) and FAp formed and retained on deciduous and permanent enamel was significantly higher in APF gel groups (p < 0.05), but no significant difference was found between 1 and 4 min (p > 0.05). The findings suggest that 1 min of APF gel application provides a similar effect on inhibition of demineralization as 4 min, for both permanent and deciduous enamel.