Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.