Nidogen and perlecan are large multifunctional basement membrane (BM) proteins conserved in all metazoa. Their high-affinity interaction, which is likely to contribute to BM assembly and function, is mediated by the central G2 domain in nidogen and the third immunoglobulin (IG)-like domain in perlecan, IG3. We have solved the crystal structure at 2.0 A resolution of the mouse nidogen-1 G2-perlecan IG3 complex. Perlecan IG3 belongs to the I-set of the IG superfamily and binds to the wall of the nidogen-1 G2 beta-barrel using beta-strands C, D and F. Nidogen-1 residues participating in the extensive interface are highly conserved, whereas the corresponding binding site on perlecan is more variable. We hypothesize that a second, as yet unidentified, activity of nidogen overlaps with perlecan binding and accounts for the unusually high degree of surface conservation in the G2 domain.