The basement membrane type IV collagen is a family composed of six genetically distinct but structurally similar polypeptide chains, alpha1-alpha6. The alpha1(IV) and alpha2(IV) chains are ubiquitous components of all BMs whereas the other four have a restricted tissue distribution. In the present study, we have analyzed the expression, distribution, and cellular origin of the alpha5(IV) and alpha6(IV) chains in the developing and adult human small intestine and in well-characterized in vitro models by indirect immunofluorescence, Western blot, and RT-PCR. We have found that in the fetal small intestine, alpha(IV) and alpha6(IV) are present in the epithelial BM and, in contrast to alpha1(IV) and alpha2(IV), are produced by both epithelial and mesenchymal cells. A distinct tissular origin for the alpha1/alpha2(IV) and alpha5/alpha6(IV) chains suggests that alpha5(IV) and alpha6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of alpha5(IV)/alpha6(IV) chain expression occurs in the adult intestine. Indeed, as compared with the fetal intestine, alpha6(IV) chain production is maintained while the expression of the alpha5(IV) chain is substantially reduced. Altered expression of the alpha5(IV) chain was also observed in the differentiating enterocytic-like Caco-2/15 cells, suggesting that in the intestinal model, the alpha5(IV) chain is subject to a regulated expression. Taken together, these observations indicate that the human intestinal epithelial BM contains up to four type IV collagen chains: the classical alpha1(IV)/alpha2(IV) chains, which originate from mesenchymal cells, and the alpha5(IV)/alpha6(IV) chains, which are of both epithelial and mesenchymal origin and have their expression regulated throughout development.