Successful endodontic therapy requires total debridement as well as complete obturation of the root canal to the cemento-dentinal junction. The goal of this study was to investigate the feasibility of using quantitative fluorescence spectroscopy for the detection and localization of pathological dentin, pulpal remnants, and microorganisms within the root canal. Specific aims were to identify: 1) characteristic excitation/emission spectra for healthy dentin, decayed dentin, enamel, and pulp; 2) the potential of specific spectral data for differentiating between these tissues; and 3) the potential of spectral data for detecting the presence and identifying four common endodontic pathogens. Fluorescence spectra were determined in the tissues of permanent human teeth, extirpated healthy and necrotic pulps, and four endodontic pathogens. Excitation/emission spectra were collected at 366nm, 405nm, and 440nm excitation. Marked differences in spectral signatures between the different tissues under investigation were observed. We postulate that the differences in fluorescence spectra of decayed vs. healthy dentin are due to the loss of mineralized tissue components and increased organic presence and water in these tissues. Pulpal tissue showed distinctly different fluorescence spectra from healthy and decayed dentin, providing a basis for differentiating between tissue categories. Each bacterial species demonstrated distinct spectral emission patterns.Ms. Sarkissian and Ms. Le are both undergraduate students at the University of California, Irvine, expected to graduate in 2005. Direct correspondence and requests for reprints to Ms. Ani Sarkissian, 123 Adams St., Glendale, CA 91205; 818-281-7956 phone; anisarkiss@aol.com.