Epithelial synthesis and secretion of basal lamina has been considered to be a general feature of various vertebrate epithelium-mesenchyme interacting systems (e.g., salivary gland, mammary gland, feather, hair, and tooth morphogenesis). It has been repeatedly assumed that embryonic ectoderm and ectodermal derivatives, such as epithelial tissues associated with tooth morphogenesis, synthesize and secrete basal lamina. Basal lamina of embryonic mouse tooth organs contain laminin, type IV collagen, glycosaminoglycans, and possibly fibronectin. Ectodermally derived epithelia produce laminin, collagens, and glycosaminoglycans but they do not appear to produce fibronectin. Mesenchyme can effect basal lamina formation in vitro by releasing mesenchyme-derived fibronectin. Theiler stage 25 molar tooth mesenchymal and epithelial tissues were enzymatically separated and cultured in chemically defined media without serum, embryonic extracts, or antibiotics for periods not exceeding 24 hr. Isolated epithelia did not reconstitute a basal lamina. Mesenchymepreconditioned media, fibronectin substrata, or addition of 10% fetal calf serum induced reconstitution of epithelium-derived basal lamina. Dental mesenchyme-preconditioned medium contained, as a major component, a protein of M(r) approximately 2.3 x 10(5) identified as fibronectin by the criteria of gelatin binding and subunit molecular weight. Fibronectin was not produced by isolated epithelia. These results support the hypothesis that basal lamina ultrastructural organization results from supramolecular interactions between epithelium-derived macromolecules (e.g., type IV collagen, proteoglycans, glycosaminoglycans, and laminin) with mesenchyme-derived cell surface fibronectin.