The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and NFIX: In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic(-/-) mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic(-/-) mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic(-/-) animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.