Ameloblastin is a tooth-specific extracellular matrix protein that is thought to play a role in enamel crystal formation in the developing dentition. The murine ameloblastin promoter functions in a cell type-specific manner and contains cis-acting elements that function both to enhance and to suppress transcription. The objective of this study was to determine whether the transcription factor Cbfa1, known to be essential for transcription of other mineralized tissue genes, is also required for ameloblastin transcription. Site-directed mutagenesis of the Cbfa1-binding site (-248 base pairs) termed osteoblast-specific element 2 (OSE2) decreased ameloblastin promoter activity by greater than 50% in ameloblast-like cells. No differences in promoter activity were observed in two other oral tissue-derived cell lines transfected with similar constructs. Nuclear factor binding to the ameloblastin promoter was also shown to be cell type-specific and was altered by site-specific mutations in the OSE2 site. Cbfa1 was specifically shown to participate in the DNA-protein complexes between nuclear factors and the ameloblastin OSE2 site by supershift electrophoretic mobility shift assays. The findings that Cbfa1 interacts with functionally important regions of the ameloblastin promoter while promoter activity is diminished in constructs containing site-directed mutations in the Cbfa1 site indicate that Cbfa1 possesses an important function in transcription of the ameloblastin gene.