To evaluate bonding penetration into different enamel substrates. Ten extracted human molars were mesiodistally sectioned. Buccal and lingual enamel surfaces were divided into four equal areas using sticky wax. The central two areas of each tooth (n = 20) were demineralized for 12 weeks using an acidic gel (pH 4.8). The lateral areas served as controls. After demineralization, ten specimens were remineralized in a saliva substitute for three weeks. An amine fluoride solution (Elmex Fluid) was applied on one half of each specimen before acid etching. After etching for 120 s, an enamel-bonding agent (Heliobond) containing 0.1% rhodamine was applied onto test and control areas, and was light cured for 60 s. Subsequently, the specimens were sectioned and tag length was determined using a confocal laser scanning microscope (CLSM). Results were statistically compared with ANOVA followed by Scheffe's and Bonferroni/Dunn post hoc tests. With a mean penetration depth of 68 ± 22 µm, tags in demineralized enamel were significantly longer than in other groups (p = 0.01). Penetration decreased significantly in remineralized areas or when fluoride was used (p = 0.01), but was still significantly deeper than in control sites (p = 0.01). Penetration of an unfilled resin into enamel was considerably influenced by the degree of dental hard tissue mineralization. Penetration was increased in demineralized enamel; however, remineralized enamel also allowed good penetration of the bonding agent. Keywords: dental material, enamel-bonding agent, penetration, enamel, confocal scanning laser microscopy, in vitro