The 50-kDa dystrophin-associated glycoprotein (50-DAG) is a component of the dystrophin-glycoprotein complex, which links the muscle cytoskeleton to the extracellular matrix. 50-DAG is specifically deficient in skeletal muscle of patients with severe childhood autosomal recessive muscular dystrophy and in skeletal and cardiac muscles of BIO 14.6 cardiomyopathic hamsters. The lack of 50-DAG leads to a disruption and dysfunction of the dystrophin-glycoprotein complex in these diseases. The cDNA encoding 50-DAG has now been cloned from rabbit skeletal muscle. The 50-DAG deduced amino acid sequence predicts a novel protein having 387 amino acids, a 17-amino acid signal sequence, one transmembrane domain, and two potential sites of N-linked glycosylation. Affinity-purified antibodies against rabbit 50-DAG fusion proteins or synthetic peptides specifically recognized a 50-kDa protein in skeletal muscle sarcolemma and the 50-kDa component of the dystrophin-glycoprotein complex. In contrast to dystroglycan, which is expressed in a wide variety of muscle and non-muscle tissues, 50-DAG is expressed only in skeletal and cardiac muscles and in selected smooth muscles. Finally, 50-DAG mRNA is present in mdx and Duchenne muscular dystrophy (DMD) muscle, indicating that the down-regulation of this protein in DMD and the mdx mouse is likely a post-translational event.