The main hard tissues of teeth are composed of dentin and enamel, synthesized by the mesenchyme-derived odontoblasts and the epithelial-derived ameloblasts, respectively. Odontoblasts are highly differentiated post-mitotic cells secreting the organic matrix of dentin throughout the life of the animal. Pathological conditions such as carious lesions and dental injuries are often lethal to the odontoblasts, which are then replaced by other pulp cells. These cells are able to differentiate into odontoblast-like cells and produce a reparative dentin. In this study we reproduced this physiological event in an in vitro culture system using pulps of human third molars. Pulp cells cultured in presence of beta-glycerophosphate formed mineralization nodules, which grew all over the culture period. The immunohistochemical study revealed that, as odontoblasts, pulp cells contributing to the nodule formation express type I collagen, osteonectin, and nestin. By the exception of nestin, these proteins are also detected in the nodules. The composition of the nodules was also analyzed by Fourier transform infrared microspectroscopy. The spectra obtained showed that both the organic and the mineral composition of the nodules have the characteristics of the human dentin and differ from those of enamel and bone. Taken together, these results show that both the molecular and the mineral characteristics of the human dentin matrix are respected in the in vitro culture conditions.