Objective: The purpose of this study was to evaluate the in vivo remineralization of the possible non-resin infiltrated hybridoid layer between the hybrid layer and the subjacent dentin substrate using nanoindentation, energy dispersive x-ray spectroscopy microanalyses (EDS), and scanning electron microscopy (SEM) technologies. Method and materials: Twenty Class V cavities were placed in healthy adult monkey teeth. Each cavity was total etched with 37% phosphoric acid for 60 seconds, rinsed, and air dispersed, and SA-Primer was applied to the collagen layer. Cavities were divided into two groups: In group 1, Protect Liner (low-viscosity resin) and Clearfil AP-X (resin composite) were placed per manufacturer's directions, and no bonding agent was placed on the acid-etched interface. In group 2, Clearfil Photobond (bonding agent) was applied, and Protect Liner and Clearfil AP-X were placed as in group 1. Teeth were observed at 7 days (control) and 6 months by nanoindentation, EDS, and SEM. Results: Six-month data showed an increased nanohardness in areas 5 µm adjacent to the demineralized or partially demineralized dentin interface. Following treatment with a conventional adhesive system on the acid-etched interface (group 2), there were increased nanohardness and calcium EDS measurements in the substrate just below the resin-dentin impregnated layer. Conclusion: Our 6-month in vivo nanoindentation and EDS data demonstrate that the non-resin infiltrated zone becomes remineralized following adhesive resin treatment.