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Dentin bonding using the total-etch method has been claimed to be technique-sensitive. The aim of this study is to examine the effect of acid-etch variations on the dentin demineralization and interfacial structure of the adhesive–dentin bond using a differential staining technique. Single Bond adhesive with 35% phosphoric acid gel was used. The occlusal one-third of the crown was removed from 60 extracted, unerupted human third molars. Smear layers were created by abrading the dentin with 600 grit SiC under water for 30 s. The prepared teeth were randomly assigned to four groups according to etching time (Group 1, 10 s; Group 2, 15 s; Group 3, 30 s; Group 4, 60 s). In each group, the etching gel was: (i) applied and spread to the dentin surface and left to stand undisturbed; (ii) applied and gently agitated during etching; (iii) applied without using dispensing tips for the syringe and left for the same period as above. After rinsing, the etched dentin was then treated with the adhesive per manufacturers' instructions. 3–5 μm thin sections of the adhesive/dentin (a/d) interface were cut with a microtome and stained with Goldner's trichrome. Stained, thin sections from each prepared tooth were imaged with light microscopy. The depth and extent of dentin demineralization, and the a/d interdiffusion zone were clearly visible by this differential staining microtechnique. The thickness of the interdiffusion zone increased as a function of etching time. However, the etchant gel application methods have a significant influence on dentin demineralization. Although agitating acid gel facilitates the penetration and etching into dentin, it should not be recommended, especially for longer etching time. These results indicated that the etching technique has a large effect on the profile of both dentin demineralization and interfacial structure.