Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibril-associated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2. Exon 24 in fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (fibrillin-1) and congenital contractural arachnodactyly (fibrillin-2). It is possible that these mutations alter the ability of fibrillin to bind MAGP-1, which may contribute to the severity of the disease.