Basement membranes, cell associated heteropolymers, are specialized extracellular matrices and are important for development and maintenance of tissue functions. An in vitro system of epithelial cells and fibroblasts co-culture was used to study the role of cell surface receptors in the assembly of a sub-epithelial basement membrane. A basement membrane was formed only in co-culture although fibroblasts alone synthesize and secret most basement membrane components. When epithelial cells were cultured singly on a type I collagen gel containing basement membrane proteins (laminin, entactin/nidogen), these proteins diffused through the gel and were concentrated in the sub- epithelial region over several days suggesting that these components assembled on selected surface receptors or existing matrix components. Blocking integrin Beta-1 of epithelial cells by a monoclonal antibody caused dramatic effects on C2BBe cell adhesion, basement membrane formation and epithelial polarity. However, the amount of laminin and entactin/nidogen in antibody treated co-culture remained the same as that in control. The results suggest that the assembly of sub- epithelial basement membrane in vivo require interaction between cell surface receptors and matrix components.