Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin γ1 chain (γ1III4) is important for kidney development and for nerve guidance. Other proteins may also bind γ1III4, although human nidogen-2 binds poorly to the mouse laminin γ1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and γ1III3-5, which both contain the γ1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial–mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin γ1 chain is dependent on epithelial–mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by α3β1 and α6β1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.