A novel member of the Krox family of proteins, designated Krox-25, was identified by screening clones from the cDNA libraries of a rat incisor and mouse embryo craniofacial tissue. Rat and mouse Krox-25 mRNAs are about 2.4 kb long, encoding 225 and 224 amino acids, respectively. Krox-25 consists of five zinc finger motifs homologous to the Drosophila Krüppel segmentation gene and also contains several consensus amino acid sequences for a protein kinase C binding domain. Northern blot analysis revealed an intense expression of Krox-25 mRNA in rat and mouse teeth, although it was expressed weakly in other tissues, including calvaria, brain, lung, thymus, kidney, and submandibular gland of mouse. In situ hybridization showed that Krox-25 mRNA began to be expressed weakly in the early odontogenic mesenchyme and primitive enamel epithelium located at the apical end of the rat incisor, and Krox-25 expression increased in the presecretory ameloblasts and became intense in the secretory ameloblasts. This expression was also similar to the results of immunohistochemistry and Western blot, especially the Krox-25 localization in the nuclei of enamel epithelial cells. These results suggest that Krox-25 plays an important role as a transcription factor for the cytodifferentiation and amelogenesis of enamel epithelium.