The epithelial-mesenchymal interface of the intestinal mucosa obviously plays an important role in supporting the mucosal architecture. Its significance for the process of migration and differentiation of the epithelial cells remains to be resolved. It consists of a basement membrane, the anchoring zone and the subepithelial connective tissue, the origin of which is unknown. We therefore established an in vitro model to study the development of the endodermal-mesenchymal interface of the fetal human and murine intestinal mucosa. The distribution of the interstitial collagens type I, III, VI and procollagen type III as well as the basement membrane components collagen type IV and laminin was investigated immunohistochemically in these fetal explant cultures. The cultures were also adapted to serum free culture conditions. It was evident that while laminin and collagen type IV could be detected in the primary intestinal epithelium, the formation of an authentic basement membrane required the presence of both the epithelial and the mesenchymal cells. Interstitial collagens and procollagen type III were produced exclusively by the mesenchymal cells. Basement membrane formation in vitro coincided with cytodifferentiation of the endodermal cells as betrayed by electron microscopy and the activity of brush border enzymes. In conclusion, the maturation of the endoderm and the formation of the subepithelial basement membrane require the intimate proximity of viable mesenchyme in vitro.