Our aim was to investigate the structural changes of dentinal tubules in specimens obtained from both hypersensitive and naturally desensitized areas in wedge-shaped defects on the same exposed cervical dentin surface of a hypersensitive tooth. A new biopsy technique that makes use of a hollow, cylindrical diamond bur was designed so that specimens from exposed root dentin of vital teeth could be obtained. Twenty-two dentin biopsy pairs were divided into two groups; one was prepared for scanning electron microscopy (SEM) and the other for microradiography (MR). Small hypersensitive areas were identified by a scratch test on exposed human dentin in vivo. SEM observation of the dentin biopsies showed that the orifices of many dentinal tubules in hypersensitive areas were open and that membranous structures appeared on the walls of dentinal tubules. In naturally desensitized areas on the same dentin surface, most of the dentinal tubules were obturated with rhombohedral crystals of all sizes; membranous structures were not observed in these tubules. These results showed that hypersensitivity occurred on the exposed dentin when most of the tubular orifices were open.