Adhesion of restorative and protective materials to dentin is an important requirement for operative and preventive dentistry. Wettability and roughness are dentin substrate conditions that are critical to establishing good adhesion. This study examined superficial and deep dentin for variations in water contact angle measurements and roughness for polished, etched, dehydrated, and rehydrated states. Superficial and deep dentin disks from 6 non-carious third molars were prepared for AFM (Atomic Force Microscope) observation, roughness measurement, and contact angle measurements following specific treatments: hydrated and polished, etched (10% H3PO4), dehydrated (desiccator for 24 hrs); and rehydrated (in water for 24 hrs). Contact angles were measured by means of the ADSA (Axisymmetric Drop Shape Analysis) technique with filtered and purified water of surface tension 72.79 ergs/cm2. The AFM was used to quantify the intertubular roughness. Mean and SD of roughness and contact angle were calculated for each dentin state, and two-way Repeated Measures ANOVA with Tukey's HSD multiple comparisons were performed at p < 0.05. Wetting and roughness both increased after etching, with roughness tending to increase further while wettability dramatically decreased after desiccation. After rehydration, water contact angle values were equivalent to those of the etched condition. Although intertubular roughness did not depend on depth, lower water contact angles were found for deep dentin. Depth and dehydration resulted in altered dentin substrates with exposed hydrophobic moieties that could interfere with bonding to hydrophilic primer coats.