Members of the transforming growth factor-beta family (e.g. TGF-beta, BMP and activin) are critical regulators of tooth morphogenesis. The basic TGF-beta signaling engine consists of a receptor complex that activates Smads and a Smad-containing complex that controls transcription of the downstream target genes. Little is known about the expression of endogenous Smads during tooth morphogenesis. Using a cRNA probe or antibody which specifically recognizes the expression of each Smad molecule, we provide a comprehensive endogenous Smad expression analysis during tooth morphogenesis. BMP signaling is transmitted through Smad1 and 5 which are first expressed within the dental lamina and later expand into condensed dental mesenchyme at the bud stage. As tooth development advances into the cap and bell stage, BMP signaling Smads are strongly localized within the inner enamel epithelium (IEE) and cranial neural crest derived dental mesenchyme (DM), indicating their critical role in regulating epithelial-mesenchyme interaction during tooth morphogenesis. Smad2 and 3 are responsible for transmitting TGF-beta/activin signaling and show unique expression patterns during tooth morphogenesis. They are localized within the nuclei of both IEE and DM, suggesting that TGF-beta-activated Smads are critical for regulating tooth development. Smad4, the common Smad, is expressed in both dental epithelium and mesenchyme throughout all stages of tooth morphogenesis. The expression of inhibitory Smads (Smad6 and 7) largely overlaps with receptor regulated Smads, indicating that negative feedback on BMP/TGF-beta signaling is critical throughout all stages of tooth morphogenesis. Our results suggest that both receptor-regulated and inhibitory Smads are important regulators of tooth morphogenesis. The selective activation of Smad, as indicated by nucleartranslocation, may suggest selective activation of different members of the TGF-beta superfamily during tooth development.