Objective: To evaluate the capability of different adhesive systems to inhibit in vitro caries formation. Method and Materials: Four materials were tested: Gluma Comfort Bond (GL; Heraeus Kulzer), Gluma Comfort Bond + Desensitizer (GL+; Heraeus Kulzer), iBond (iB; Heraeus Kulzer), and One-up Bond F (OUB; Tokuyama). Bovine roots were ground to obtain flat mesial and distal dentin surfaces. Nail varnish was applied to the entire root surface except for two 10 mm X 2 mm windows on the flattened surfaces. The adhesives were applied to the exposed areas according to the manufacturers' instructions. The roots were incubated at 37°C for 1 week in a suspension of StreptococcuS mutans and Lactobacillus acidophilus. Control groups either received no root surface treatment or were etched with 37% phosphoric acid (without immersion in the bacterial suspension). Specimens were sectioned perpendicular to the long axis of the roots, stained for 24 hours with Rhodamine B, and analyzed with confocal laser microscopy. The data were analyzed by 1-way ANOVA and Fisher's PLSD test at 95% confidence level. Results: Lesions in the OUB, iB, and GL+ groups were significantly shallower than those in the no-treatment group. Acid etching did not produce any measurable lesions. Mean lesion depth in the GL group was similar to that in the no-treatment group. There was no significant difference between mean lesion depths in the GL+, GL, and iB groups. Conclusion: Fluoride- and glutaraldehyde-containing adhesive systems might be an aid in root caries prevention. Keywords: adhesive, inhibition, in vitro, Lactobacillus acidophilus, root caries, Streptococcus mutans