This study evaluated the influence of sub-ablative Er,Cr:YSGG laser and topical fluoride application on incipient caries development in vitro. One hundred and sixty human enamel slabs were ramdomly divided into eight equal groups: (1) untreated (control); (2???4) irradiated with Er,Cr:YSGG laser at 0.25, 0.50 and 0.75 W, respectively; (5) treated with acidulated phosphate fluoride (APF; 1.23% F) for 4 min; (6???8) pre-irradiated with Er,Cr:YSGG laser at 0.25, 0.50 and 0.75 W, respectively and subjected to APF application. All groups were submitted to pH-cycling simulating a cariogenic challenge, and after 10 cycles mineral loss ( ?? Z) was measured in enamel. Calcium, inorganic phosphorus (P i ) and fluoride concentrations were also measured in the demineralizing and remineralizing pH-cycling solutions. Data were analyzed by ANOVA ( ?? = 0.05). No differences in ?? Z were found between laser irradiation at 0.25 W, 0.50 W and controls, while after laser irradiation at 0.75 W (Group 4) and application of APF (Group 5) there was a significant decrease in ?? Z. Laser irradiation at 0.50 and 0.75 W prior to APF application there was a significantly lower ?? Z than in other groups. A significant increase in Ca and P i concentrations was observed in demineralizing solutions compared to the initial concentrations, but no significant changes in Ca and P i concentrations were detected between any groups. A significant decrease in fluoride concentration was observed in pH-cycling solutions of control and 0.25 W laser groups, while the association of laser at 0.50 and 0.75 W with fluoride promoted an increase of fluoride content in remineralizing solutions. In conclusion, Er,Cr:YSGG laser irradiation at 0.50 and 0.75 W, associated with fluoride, seems to be a good alternative for reducing enamel demineralization.Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP)FAPESP: 04/02229-6