Alpha- and beta-dystroglycan form a heteromeric transmembrane complex linking the extracellular matrix to the cytoskeleton. In muscle beta-dystroglycan interacts with dystrophin on the inside of the cell and with alpha-dystroglycan, which binds the extracellular matrix protein laminin, on the outside. Dystroglycan is expressed not only in muscle but also in other tissues. We cloned beta-dystroglycan from rabbit brain by RT-PCR and expressed deletion mutants of the beta-dystroglycan cytoplasmic domain as GST-fusion proteins. We identified the dystrophin binding region on beta-dystroglycan by protein overlay and co-precipitation assays with skeletal muscle dystrophin and recombinant apo-dystrophin I. We demonstrate that the beta-dystroglycan carboxyl terminus interacts with dystrophin and that the binding site is restricted to the last 20 amino acids. Our data also suggest that the region adjacent to the beta-dystroglycan transmembrane domain might modulate beta-dystroglycan-dystrophin interaction.