Background and Purpose: Topical application of fluoride onto the dental enamel surface mainly induces the formation of alkali-soluble fluoride, described as a calcium fluoride or calcium fluoride-like material (CaF2). It is believed that calcium fluoride acts as a pH-controlled fluoride-releasing reservoir. The aim of this study was to quantify, in vitro, the formation of alkali-soluble fluoride after application of amine fluoride solution with different fluoride content and different frequency of use on human dental enamel. Material and Methods: Four enamel slabs were cut from 11 impacted third molars and randomly divided into four groups, One of them served as the unfluoridated control (D). Each of the three groups of slabs was shaken for 3 min in the amine fluoride solution of different fluoride concentration with pH of 3.8; group A: 1% F twice a day; group B: 0.5% F three times a day and group C. 0.25% F four times a day. The treatments were repeated during three consecutive days, The formation of the alkali-soluble fluoride was analyzed chemically, Results: The amount of alkali-soluble fluoride (mug/cm(2)+/-SD) was significantly higher in group C (115.77+/-59.98) than in groups A (56.17+/-48.95) and B (71.69+/-37.03). No significant difference (p > 0.05) was found between A and B groups. The amount of alkali-soluble fluoride on the enamel increased with the time of exposure despite the lower content of fluoride. Conclusion: The results show that increased frequency of fluoride applications even with a lower fluoride content may result in the formation of more calcium fluoride.