Although the individual cariostatic effects of laser and fluoride have been shown, the combined effect of CO(2) laser and fluoride on root demineralization remains uncertain and was the main aim of this study. By using a pH-cycling system and Polarized Light Microscopy, we demonstrated the synergistic effect of fluoride combined with CO(2) laser treatment on reducing root demineralization. The mean lesion depths (in microm) for each group were 160 +/- 14 (Control), 113 +/- 8 (Laser treatment alone), 111 +/- 6 (Fluoride treatment alone), and 25 +/- 7 (Fluoride followed by laser treatment). A significant laser-enhanced fluoride uptake, characterized by the ToF-SIMS analysis, was revealed by the 37% and 400% increments in loosely and firmly bound fluorides (both p < 0.002) in laser-irradiated areas, compared with the non-irradiated controls. In conclusion, there is a significant synergistic effect of combined CO(2) laser and fluoride treatment on the inhibition of root demineralization, possibly due to laser-enhanced fluoride uptake in the root.