OBJECTIVE To evaluate the effect of time of fluoride application gel, acidulated or neutral, on in vitro enamel resistance to demineralization and fluoride uptake. METHODS One hundred and ninety-two human enamel blocks were used in this study and 144 were treated with fluoride gel, acidulated or neutral, for 1 or 4 minutes. Ninety-six blocks treated with fluoride and 24 control blocks were submitted to a high cariogenic challenge. After the pH-cycling, enamel demineralization was assessed by surface and cross-sectional microhardness. Fluoride in the enamel blocks was also determined after removing an enamel layer by etching acid. RESULTS Acidulated fluoride gel formed more fluoride in enamel than neutral gel (P<0.05), and it was also more efficient in reducing the demineralization of the enamel blocks submitted to a cariogenic challenge than the neutral one (P<0.05). It was found that the time of application was significant in terms of fluoride uptake, but it did not render the enamel more resistant to demineralization.