In a new culture-conditioning system of agar-coated mesenchyme of isolated incisor dental papillae, dentinogenesis has been induced adjacent to an agar substratum that functions as a foothold for cell immobilisation. To elucidate the role of the basement membrane (BM) in dentinogenesis, we have examined the way in which dentinogenesis depends upon BM components or transforming growth factor (TGF)-beta (1) in this system. At the mesenchymal-epithelial junction of odontogenic organs (cut incisor tooth germs), TGF-beta (1) visibly increased in the BM during incubation. In isolated dental papillae, BM components were synthesised and deposited at aligned peripheral cells of the explants, together with an increasing amount of TGF-beta (1). These components were not assembled into extracellular matrix (ECM)-absorbed agar adjacent to explants, although dentinogenesis proceeded in the presence of pericellular BM components associated with TGF-beta (1). When signalling via TGF-beta type II receptors was blocked, neither ECM production nor dentinogenesis was observed but explants partially detached from the agar surface, presumably as a result of the suppressed production of ECM, since attachment was retained by pre-coating explants with artificial matrices. Rescue experiments showed that TGF-beta (1) regulated dentinogenesis through ECM production. With regard to BM components, inducible dentinogenesis was Arg-Cly-Asp (RGD)-dependent. Thus, pericellular BM components associated with TGF-beta (1) and an ECM-absorbed agar substratum, which affects dentinogenesis, synergistically play a role similar to that of BM components in vivo. The BM therefore serves as a structural meshwork that acts as a foothold for cell immobilisation; its components act as ligands for RGD-dependent cell adhesion and it stores TGF-beta (1), which regulates ECM production.