The surface component β-dystroglycan is a member of the dystrophin–glycoprotein complex providing a trans-sarcolemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin- α2. Although abnormalities in this complex are involved in the pathophysiology of various neuromuscular disorders, little is known about the organization of dystrophin-associated glycoproteins in diaphragm and brain. We therefore investigated the oligomerization of β-dystroglycan and its connection with the most abundant dystrophin homologues in these two tissues. Employing detergent solubilization and alkaline extraction procedures of native membranes, it was confirmed that β-dystroglycan behaves like an integral surface molecule as predicted by its cDNA sequence. Immunoblot analysis following chemical crosslinking of native membranes showed that β-dystroglycan has a tendency to form high-molecular-mass complexes. Within these crosslinkable complexes, immuno-reactive overlaps were observed between β-dystroglycan, α-dystroglycan, laminin and 427 kDa dystrophin in diaphragm and skeletal muscle. In synaptosomes, the major brain dystrophin isoform Dp116 also exhibited an immuno-reactive overlap with members of the dystroglycan complex. These findings demonstrate that β-dystroglycan does not exist as a monomer in native membranes and imply that certain dystrophin isoforms and dystrophin-associated components interact with this surface protein in diaphragm and brain as has been previously shown for skeletal and heart muscle.