The alpha-dystroglycan binding properties of laminins extracted from fully differentiated skeletal muscle were characterized. We observed that the laminins expressed predominantly in normal adult rat or mouse skeletal muscle bound alpha-dystroglycan in a Ca2+-dependent, ionic strength-sensitive, but heparin-insensitive manner as we had observed previously with purified placental merosin (Pall, E. A., Bolton, K. M., and Ervasti, J. M. 1996 J. Biol. Chem. 271, 3817-3821). Rat skeletal muscle laminins partially purified by heparin-agarose affinity chromatography also bound alpha-dystroglycan without sensitivity to heparin. We also confirm previous studies of dystrophic dy/dy mouse skeletal muscle showing that the alpha2 chain of merosin is reduced markedly and that the laminin alpha1 chain is not up-regulated detectably. However, we further observed a quantitative decrease in the expression of laminin beta/gamma chain immunoreactivity in alpha2 chain-deficient dy/dy skeletal muscle and reduced alpha-dystroglycan binding activity in laminin extracts from dy/dy muscle. Most interestingly, the alpha-dystroglycan binding activity of residual laminins expressed in merosin-deficient dy/dy skeletal muscle was inhibited dramatically (69 +/- 19%) by heparin. These results identify a potentially important biochemical difference between the laminins expressed in normal and dy/dy skeletal muscle which may provide a molecular basis for the inability of other laminin variants to compensate fully for the deficiency of merosin in some forms of muscular dystrophy.