This study evaluated the immunohistochemical labeling pattern of dentin collagen fibrils within hybrid layers created by different bonding systems using high resolution SEM. Four different adhesive materials, including self-etching and total-etching systems, were examined: Prime & Bond NT, OptiBond SOLO Plus, Single Bond and Clearfil Protect Bond. All materials were applied to the dentin of extracted human third molars. After cutting the bonded specimens transversely, an anti-collagen type I antibody was incubated on the surface of the dentin-adhesive interface and gold-conjugated secondary antibody was applied to reveal collagen labeling under high resolution SEM. The hybrid layers showed a significant number of collagen fibrils embedded in the resin matrix. The presence of exposed dentin collagen fibrils, as determined by positive labeling with an anti-type I collagen monoclonal antibody, is considered an indication of the presence of incompletely embedded fibrils and, thus, the quality of dentin matrix hybridization. The hybrid layers produced by total-etching systems showed higher labeling compared to those produced by the self-etching system. Positive collagen labeling was also found along the resin tags produced by total-etching adhesive systems.