The dentinoenamel junction (DEJ) is a complex and poorly defined structure that unites the brittle overlying enamel with the dentin that forms the bulk of the tooth. In addition, this structure appears to confer excellent toughness and crack deflecting properties to the tooth, and has drawn considerable interest as a biomimetic model of a structure uniting dissimilar materials. This work sought to characterize the nanomechanical properties in the region of the DEJ using modified AFM based nanoindentation to determine nanohardness and elastic modulus. Lines of indentations traversing the DEJ were made at 1-2 microm intervals from the dentin to enamel along three directions on polished sagittal sections from three third molars. Nanohardness and elastic modulus rose steadily across the DEJ from bulk dentin to enamel. DEJ width was estimated by local polynomial regression fits for each sample and location of the mechanical property curves for the data gradient from enamel to dentin, and gave a mean value of 11.8 microm, which did not vary significantly with intratooth location or among teeth. Nanoindentation was also used to initiate cracks in the DEJ region. In agreement with prior work, it was difficult to initiate cracks that traversed the DEJ, or to produce cracks in the dentin. The fracture toughness values for enamel of 0.6-0.9 MPa . m(1/2) were in good agreement with recent microindentation fracture results. Our results suggest that the DEJ displays a gradient in structure and that nanoindenation methods show promise for further understanding its structure and function. Copyright 2000 John Wiley & Sons, Inc.