To investigate the extent of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action and its relationships to calbindin gene expression in mineralized tissues, we have analyzed rat incisors with different probes, including a vitamin D receptor (VDR) antibody and specific cDNAs to rat calbindin-D9K and calbindin-D28K. Developmental and hormonal controls of calbindin gene expression were investigated by Northern blot analysis of ameloblast and odontoblast mRNA. Distribution and hormone-induced changes of VDR were also studied by light microscopic immunocytochemistry. A differential tissue- and stage-specific expression of the calbindin genes was observed in microdissected portions of the continuously erupting incisor. The two calbindins were expressed in ameloblasts, whereas only calbindin-D28K was expressed in odontoblasts. Moreover, in ameloblasts, expression of calbindin-D28K preceded that of calbindin-D9K. Immunoreactivity for VDR was present in all progenitor cells and progressively decreased during the differentiation process, whereas, in differentiated tissues, a hormonal upregulation was restricted to hard tissue-forming cells, i.e., ameloblasts and odontoblasts. Furthermore, calbindin gene expression appeared to be regulated by 1,25(OH)2D3. Taken together, these data indicate that ameloblasts and odontoblasts are target cells for 1,25(OH)2D3 and provide the first insights into the hormonal control of tooth genes during development.