Background: It remains uncertain as to whether or not CO 2 laser is able to hinder demineralization of enamel. The possibility to use bovine instead of human teeth on anticariogenic studies with laser has not yet been determined. Purpose: To compare the ability of CO 2 laser and fluoride to inhibit caries-like lesions in human enamel and to test whether a similar pattern of response would hold for bovine enamel. Study Design: Ninety-six enamel slabs (2 3 2 3 4 mm) (48 from bovine and 48 from human teeth) were randomly distributed according to surface treatment (n 5 12): CO 2 laser, 5% sodium fluoride varnish (FV), 1.23% acidulated phosphate fluoride (APF) gel, or no treatment (control). Specimens were subjected to a 14-day in vitro cariogenic challenge. Microhardness (SMH) was measured at 30 lm from the surface. For ultrastructural analysis, additional 20 slabs of each substrate (n 5 5) received the same treatment described earlier and were analyzed by SEM. Results: ANOVA and Tukey test ascertained that CO 2 laser promoted the least mineral loss (SMH 5 252 a ). Treatment with FV resulted in the second highest values (207 b ), which was followed by APF (172 c ). Untreated specimens performed the worst (154 d ). SEM showed no qualitative difference between human and bovine teeth. APF and control groups exhibited surfaces covered by the smear layer. A granulate precipitate were verified on FV group and fusion of enamel crystals were observed on lased-specimens. Conclusions: CO 2 laser may control caries progression more efficiently than fluoride sources and bovine teeth may be a suitable substitute for human teeth in studies of this nature.