Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.