1) 76 strains belonging to 17 species or bacterial groups were tested in respect of pigmentation of the small piece of the tooth placed in 1% glucose broth. It was found that the organisms which produced acid from glucose could stain deeper depending on their pH level of the broth reached during 20 days incubation at 37°C.2) One of the determinate factors for the pigmentation was found in the meat extract which consist of the glucose broth and it was removed after absorption by activated charcoal. The second determinant was considered to be releasing of reactive groups of the tooth which is inactive under physiological circumstances.3) The activation of such reactive groups might be brought by either lowering pH of the medium or addition of chelating agent, used Na2-EDTA in this study, in alkaline reaction. Thus it might be the removal of calcium blocking the reactive groups of some organic moiety of the tooth.4) In addition to the meat extract, browning substances were also found in yeast extract, soy bean sauce, autoclaved pepton solution containing humin, caramelized glucose solution and neutralized pyruvic acid solution. Exogenous or dietary pigments may also play some role in the browning of the carious process.5) The fact that pigmentation occured in the caramelized glucose solution in alkaline reaction is most interesting in this study. Formation of Ca-saccarate may cause releasing the reactive groups of the tooth.