OBJECTIVE The purpose of this work was to compare fluoride release from three orthodontic adhesives and fluoride penetration into the enamel surface. METHODS A total of 156 extracted human premolar teeth were randomly assigned to three experimental groups and one control group (without bonding) with 39 teeth per group. Brackets were bonded to teeth using Fuji Ortho LC®, Illuminate®, or Light Bond®. The amount of fluoride released (ppm) into artificial saliva was measured by a fluoride ion-selective electrode connected to an ion analyzer on days 1, 3, 7, and 30. Fluoride penetration was investigated after 1, 2, and 3 months; 13 teeth of each group were randomly selected at every period of study and sectioned across the center of the bracket. The surface of the cross-section was studied under the scanning electron microscope, and the fluoride concentration (weight%) at 1, 2, and 3 µm below the outer enamel surface was determined by energy-dispersive X-ray microanalysis. RESULTS On days 1, 3, 7, and 30, the mean cumulative fluoride release from the three orthodontic adhesives were significantly different (p < 0.05). Illuminate® released the greatest fluoride, followed by Fuji Ortho LC® and Light Bond®. After 1, 2, and 3 months, fluoride penetration into enamel was only found from Fuji Ortho LC®. The fluoride concentration decreased with depth but there were no significant differences (p > 0.05) over time at all depths. CONCLUSIONS The in vitro study indicated that fluoride release is a common property of the three fluoride-releasing orthodontic adhesives: Illuminate®, Fuji Ortho LC®, and Light Bond®. However, detectable fluoride penetration is a specific property of Fuji Ortho LC®. Further clinical studies should be undertaken to investigate the benefit of the two adhesives Illuminate® and Fuji Ortho LC® on protection of enamel demineralization.