In this review we describe applications of fluorescence microscopy to investigations of dental hard tissue. Many studies have shown that human teeth show autofluorescence and several applications of fluorescence microscopy have been reported, although the chemical nature of autofluorescence remains unknown. However, most applications are based on a static measurement mode, which has inherent limitations. These limitations can be overcome by the use of time-resolved fluorescence microscopy. Therefore, we attempted to combine ordinary and time-resolved fluorescence microscopy to study human dentine. Using stationary measurements, blue fluorescence with a peak of 440 +/- 10 nm and a width of approximately 100 nm was observed. Increases in fluorescence intensity were found to be dependent upon age and temperature, regardless of tooth type or gender. The results suggest that the aging mechanism for crown dentine differs from that for root dentine and that the observed increase in fluorescence intensity is partially due to a temperature-dependent physicochemical mechanism. We speculate that fluorescence microscopy will become a useful tool for analysis of dental hard tissue in several fields.