Objective: Dental adhesion has become one of the most challenging topics of restorative dentistry. Acids are now applied on dentin to improve adhesion. If the acid penetrates deeper than the adhesive resin subsequently applied, an area of vulnerable collagen fibers may weaken the bonding. This in vitro study was designed to evaluate the effect of etching time on the depth of dentin demineralization, using Field Emission Scanning Electron Microscopy. The null hypothesis tested was that the depth of dentin demineralization would not vary proportionally with etching time. Method and materials: Twenty-one dentin disks were obtained from extracted human molars by diamond saw sectioning. The specimens were equally assigned to 7 different etching times (n = 3). The dentin surfaces were etched with 35% phosphoric acid, fixed, dehydrated, and dried. The specimens were observed using a Field Emission Scanning Electron Microscope. The morphological appearance of the dentin surfaces was compared both from an occlusal view and from a lateral view. The depth of demineralization of intertubular dentin was registered for all specimens. Results: For an etching time of 5 seconds, the mean depth of demineralization was 1.1 µm. For an etching time of 120 seconds, the mean depth of dentin demineralization was 8.1 µm. Conclusion: Although there was a significant correlation between etching time and depth of intertubular penetration, the depth of phosphoric acid penetration into dentin was not proportionally correlated to the respective etching time. When the acid was replenished with fresh etchant after 60 seconds, the depth of penetration increased substantially.