The laminin binding properties of α-dystroglycan purified from rabbit skeletal muscle membranes were examined. In a solid phase microtiter assay, 125I-laminin (laminin-1) bound to purified α-dystroglycan in a specific and saturable manner with a half-maximal concentration of 8 nM. The binding of 125I-α-dystroglycan to native laminin and merosin (a mixture of laminin-2 and -4) was also compared using the solid phase assay. The absolute binding of 125I-α-dystroglycan to laminin (6955 ± 250 cpm/well) was similar to that measured for merosin (7440 ± 970 cpm/well). However, inclusion of 1 mg/ml heparin in the incubation medium inhibited 125I-α- dystroglycan binding to laminin by 84 ± 4.3% but inhibited 125I-α- dystroglycan binding to merosin by only 17 ± 5.2%. Similar results were obtained with heparan sulfate, while de-N-sulfated heparin, hyaluronic acid, and chondroitin sulfate had no differential effect. These results were confirmed by iodinated laminin and merosin overlay of electrophoretically separated and blotted dystrophin-glycoprotein complex. In contrast to the results obtained with skeletal muscle α-dystroglycan, both laminin and merosin binding to purified brain α-dystroglycan was significantly inhibited by heparin. Our data support the possibility that one or more heparan sulfate proteoglycans may specifically modulate the interaction of α-dystroglycan with different extracellular matrix proteins in skeletal muscle.