Tissue reflectance spectrophotometry (TRS) can estimate hemoglobin concentration (Hb index) and oxygen saturation of hemoglobin (apparent SO2) in tissues. Reflectance spectra were measured in a gingiva-model and in human gingival biopsies by TRS. The gingiva-model was composed of human red cells and white gel in a cuvette placed on a black or white surface. The distance from the bottom surface of the cuvette to the optical probe was decreased from 5.0 to 0.5 mm. The Hb index in the gingiva-model on the black surface was constant before decreasing at less than a 3.0 mm distance. However, the Hb index in the gingiva-model on the white surface showed a decrease at less than a 1.5 mm distance. Apparent SO2 was constant at more than a 1.0 mm distance. Reflectance spectra were measured from human gingival tissue preparations, by slicing them off with microtome and making them less thick each time. Changes in reflectance spectra were observed in less than 1.0-1.5 mm thick specimens. These results suggest that the sensitivity in TRS measurement may exist within 1.0-1.5 mm from the gingival surface, and the limit of depth at which the TRS can measure may be within 3.0 mm.