Objectives: To evaluate the fluoride releasing effect of a dentin bonding material over time on nanoleakage. Material and Methods: Standardized class-V cavities were prepared in 60 extracted human molars. 30 cavities were filled with composite (Spectrum) using the Prime&Bond NT bonding system (containing cetylamine hydrofluoride). 30 teeth were filled with the same composite and an experimental Prime&Bond NT without fluoride content. Prior to the examination 15 teeth per group were stored for 24 h in water and the remaining 15 teeth per group were stored for 6 month. After storage the teeth were exposed to a 1% rhodamin B solution for 24 h at 20°C, rinsed in water for 60 s and sectioned parallel to the tooth axis, dividing the restorations into two parts. A confocal laser scanning microscope (CLSM) was used in fluorescence mode to visualize a layer 10 µm below the prepared surface. The lengths of the penetrated pathways were measured. Results: High resolution CLSM images demonstrated a penetration pathway which was located at the junction between hybrid layer and dentin. Storage for 24 h resulted in penetration lengths of 53 ± 59 µm. After 6 month the penetration length was 74 ± 40 µm for the fluoride releasing bonding agent and 75 ± 52 µm for the experimental bond without fluoride content. Storage for 6 month slightly increased dye penetration lengths, but the difference is not statistically significant (p=0.09 with fluoride release and p=0.11 without fluoride release; Mann-Withney U-test). It is concluded that the penetrations observed are a result of nanoleakage and the fluoride release has no effect on nanoleakage. Keywords: dentin bonding agent, nanoleakage, fluoride