This study evaluated the effect of Er,Cr:YSGG laser irradiation and professional fluoride application on enamel demineralization and on fluoride formation and retention. In a blind in vitro study, 264 human enamel slabs were distributed into 8 groups: G1--untreated; G2--treated with acidulated phosphate fluoride gel (APF gel, 1.23% F) for 4 min; G3, G4 and G5--irradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively; G6, G7 and G8--preirradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively, and subjected to APF gel application. Twenty slabs of each group were submitted to a pH-cycling regimen, and enamel demineralization was evaluated in 10 slabs of each group. In the other 10 slabs, CaF2-like material was determined. To evaluate F formed, 10 additional slabs of each group, not subjected to the pH cycling, were submitted to analysis of CaF2-like material and fluorapatite, while the other 3 slabs of each group were evaluated by scanning electron microscopy. The F content was also measured in all pH-cycling solutions. Laser at 8.5 J/cm2 and APF treatment reduced enamel demineralization compared to the control (p < 0.05), but the combination of these treatments was not more efficient than their isolated effect. A higher concentration of retained CaF2-like material was found in laser groups followed by APF in comparison with the APF gel treatment group. The findings suggest that laser treatment at 8.5 J/cm2 was able to decrease hardness loss, even though no additive effect with APF was observed. In addition, laser treatment increased the formation and retention of CaF2 on dental enamel.