Six extracted human teeth with naturally-formed neglected stain were analyzed for chemical constituents using wavelength dispersive spectrometry (WDS), an electron microprobe technique. Spatial distribution, within a few microm resolution, of the compositional elements was obtained by line and map analyses, which provided relative concentrations of the elements in stain-enamel complex. Absolute concentrations at different locations across a specimen were obtained from quantitative analysis. Results showed that these neglected stains were highly calcified and contained a significant amount of organic matter (C, N, O, S), with traces of Fe and Cu. The tooth surface underneath the stain layer could be easily distinguished by the higher Ca and P content, as well as by finite amounts of C and S. Corresponding areas of high concentrations between S and Fe/Cu were observed, which suggested the complex of sulfur and metal ions as possible color-forming species. S was found to diffuse into surface enamel in the range of 10 microm.