Previous studies have shown that bleaching treatment may be efficient in both enamel and dentin, but it is still unknown how much the subsurface dentin contributes to the color change of teeth. This in vitro study evaluated the whitening effect of different external bleaching agents on enamel-dentin slabs and subsurface dentin. Ninety bovine teeth were distributed among six groups (A, Opalescence 10%; B, Opalescence PF 15%; C, Opalescence Quick; D, Opalescence Extra Boost; E, Rapid White; F, Whitestrips). Two enamel-dentin specimens were prepared from the labial surface of each teeth. In one of the specimens enamel was removed, resulting in a dentin (CD) disc of 1 mm high. The labial and the pulpal sides of the second specimen were ground until the remaining enamel and dentin layers of the enamel-dentin sample (ED) were 1 mm each. Whitening treatment of the ED specimens was performed according to manufacturers' instructions. Pre- and posttreatment Lab values of ED samples were analyzed using CIE-Lab. Baseline Lab values of dentin were analyzed by evaluation of the CD specimen. Finally, enamel of the ED specimens was removed and color change of the exposed dentin (D) was recorded. For all treatment agents significant color changes (DeltaE) were observed for enamel-dentin samples and subsurface dentin specimens compared to controls. In groups A-D DeltaE was significantly higher in dentin than enamel-dentin. Furthermore, L and b values of bleached enamel-dentin and subsurface dentin samples differed significantly from baseline. Treatment with the tested external whitening bleaching agents resulted in color change of both enamel-dentin and subsurface dentin samples. The results indicate that color change of treated teeth might be highly influenced by color change of the subsurface dentin.