Objective: To compare, using transmission electron microscopy (TEM), the ultrastructure of the hybrid layers formed beneath a one-bottle adhesive system using the total-etch technique with that of a self-etching system. Method and materials: Occlusal cavity preparations were made in vivo in 18 human premolars and randomly appointed to three groups (n = 6), according to the following bonding procedures: (1) OptiBond Solo, a single-bottle adhesive system, was applied following 15 seconds etching with 37% phosphoric acid and rinsing; (2) Clearfil Liner Bond 2V, a self-etching adhesive system was applied; and (3) no bonding material was used, with the untreated smear layer acting as a control. The cavities were then filled with resin composite restorations. Results: In group 1, dense resin tags obturated the tubules. A layer of inorganic silicon microgranules formed at the top of the interdiffusion zone, and below this was a zone of loosely arranged collagen fibers. Toward the base, there was a more dense accumulation of hydroxyapatite crystals. In group 2, dense resin tags obturated the tubular orifices. Collagen fibers were densely compacted within monomer material. Toward the base, hydroxyapatite crystals were observed between collagen bundles. In group 3, a rough fragmented smear layer covered the dentinal floor and occluded the tubules. Conclusion: Irregularities occurred in the coronal zone of the hybrid layer in both systems. In the one-bottle system, this was attributed to the etching technique, while in the case of the self-etching system, this was attributed to the resin materials. These irregularities are potential sites for debonding in the clinical situation.