Target cells for 1,25(OH)2 vitamin D3 metabolites are identified in developing rodent teeth by the use of thaw-mount autoradiography. Following the injection of [26, 27-3H]-1,25(OH)2 vitamin D3 into 18-day- and 20-day-old fetal rats and neonatal mice, nuclear concentration of radioactivity is found in different cell types. In incisors of both animal groups, strong nuclear labeling is present predominantly in pulp cells, while relatively weakly labeled cells are found in the layers of odontoblasts, ameloblasts, and stratum intermedium. In molars, nuclear labeling is absent in fetal rats, but is present in 2-day-old neonates in pulp cells and cells in the layers of stratum intermedium of the first molars, but not in the second molars. The absence of labeled pulp cells in the progenitor regions of incisors and in molars of 20-day-old fetal rats, and differential ontogenic appearance of labeled pulp cells in molars, indicates that there is a critical period of receptor emergence. The finding that labeled pulp cells exist in the regions of incisors and molars where secretory odontoblasts are present suggests that nuclear uptake of 1,25(OH)2 vitamin D3 is related to cell maturation and differentiation, and topographically related to the formation of dentin. The results further suggest that, in contrast to bone, the predominant effect of 1,25(OH)2 vitamin D3 is not on tooth cells which are directly involved in the formation of calcified tissue, i.e., ameloblasts and odontoblasts, but rather on supporting tissues such as pulp cells and stratum intermedium.