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This study compared the thickness of the hybrid layer formed using Scotchbond Multi-Purpose Plus, Single Bond 2, Prime & Bond 2.1 and Xeno III on a dentin surface prepared with a diamond bur in a high speed handpiece or prepared with an Er:YAG laser used with two parameters of pulse energy (200 and 400 mJ) and two parameters of frequency (4 and 6 Hz). Flat dentin surfaces obtained from 20 human third molars were treated with the two methods and were then prepared with the dentin adhesive systems according to the manufacturers' instructions. After a layer of composite was applied, the specimens were sectioned, flattened, polished and prepared for Scanning Electronic Microscopy observation. Five different measurements of the hybrid layer thickness were obtained along the bonded surface in each specimen. The results were statistically analyzed using Analysis of Variance and Student-Newman-Keuls tests (p < or = 0.05). When analyzing the hybrid layer thickness and comparing the cavity preparation method, four groups were formed: Group I (diamond bur) > Group II (Laser 200 mJ/4 Hz) = Group III (Laser 200 mJ/6 Hz) > Group IV (Laser 400 mJ/4 Hz) > Group V (Laser 400 mJ/6 Hz). When comparing the dentin adhesive systems, there were no statistically significant differences. These results showed that the four tested dentin adhesive systems produced a 2.90 +/- 1.71 microm hybrid layer in dentin prepared with a diamond bur. This hybrid layer was regular and routinely found. In the laser groups, the dentin adhesive systems produced hybrid layers ranging from 0.41 +/- 1.00 microm to 2.06 +/- 2.49 microm, which were very irregular and not routinely found. It was also concluded that the Er:YAG laser, with the parameters used in this experiment, has a negative influence on the formation of a hybrid layer and cavity preparation methods influence formation of the hybrid layer.