When brain proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose are probe with 125I-labeled laminin, a single broad band of approximately 120 kDa binds laminin specifically. We show here by two-dimensional electrophoresis and protein microsequencing that this band consists of two distinct laminin-binding proteins. One of these is the amyloid precursor protein. The other, laminin-binding protein (LBP) 120, is closely related to the dystrophin-associated glycoprotein, dystroglycan (156 kDa); 5 peptides from purified bovine brain LBP120, ranging in size from 7 to 19 residues, are up to 100% identical to the predicted amino acid sequence of muscle dystroglycan (ibraghimov-Beskrovanaya, O., Ervasti, J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., and Campbell, K. P. (1992) Nature 355, 696-702). These protein microsequence data support the data of Ibraghimov-Beskrovnaya et al., which suggest that the dystroglycan precursor is processed into 120/156- and 43-kDa proteins. Moreover, the data suggest a revision in the position of the proposed cleavage site of the precursor. The glycosylation and extracellular localization of LBP120/dystroglycan are consistent with it being a cell surface laminin receptor. LBP120/dystroglycan, either as a native protein, or following SDS-PAGE and transfer to nitrocellulose, binds with high affinity (Kd = 90 nM) to a proteolytic fragment of laminin (E3) containing the major heparin binding domain. This binding is Ca(2+)-dependent and inhibited by low concentrations of heparin. Thus, LBP120/dystroglycan is a major non-integrin laminin receptor whose high affinity interaction with laminin may reflect a structural role in brain and muscle.