Introduction: Study aimed to establish the effect of calcium lactate enamel pretreatment related to different fluoride types and concentrations on enamel uptake of alkali-soluble fluorides. Materials and methods: In a blind and randomized in vitro study, total of 60 teeth are used. First 30 teeth, cut and randomly allocated into one of the following treatment: A) Calcium lactate pretreated followed by 3 different fluoride solution; B) ”Fluoride only” group, slabs are treated with 3 different fluoride solution; C) “Calcium only” group, slabs treated with calcium lactate solution; D) slabs are treated with deionized water (negative control group). The next 30 teeth underwent all above described group procedures, but treated with lower fluoride concentrations. Fluoride was extracted from enamel using 1M KOH solution and analyzed using fluoride ion-specific electrode. Wilcox Matched Pairs test and Friedman ANOVA analyzed the effect of substrate and treatments. Results: The findings revealed that slabs treated with NaF following calcium lactate pretreatment exhibited significantly greater enamel uptake of alkali-soluble fluoride compared to other substrates. This significant effect is not observed at lower fluoride concentrations. Conclusion: The study demonstrates that pretreatment with calcium lactate followed by treatment with NaF at 226 ppm F significantly enhances the uptake of alkali-soluble fluoride in enamel compared to other fluoride type.