The dystrophin-glycoprotein complex is a multisubunit protein complex that spans the sarcolemma and forms a link between the subsarcolemmal cytoskeleton and the extracellular matrix. Primary mutations in the genes encoding the proteins of this complex are associated with several forms of muscular dystrophy. Here we report the cloning and characterization of sarcospan, a unique 25-kDa member of this complex. Topology algorithms predict that sarcospan contains four transmembrane spanning helices with both N- and C-terminal domains located intracellularly. Phylogenetic analysis reveals that sarcospan's arrangement in the membrane as well as its primary sequence are similar to that of the tetraspan superfamily of proteins. Sarcospan co-localizes and co-purifies with the dystrophin-glycoprotein complex, demonstrating that it is an integral component of the complex. We also show that sarcospan expression is dramatically reduced in muscle from patients with Duchenne muscular dystrophy. This suggests that localization of sarcospan to the membrane is dependent on proper dystrophin expression. The gene encoding sarcospan maps to human chromosome 12p11.2, which falls within the genetic locus for congenital fibrosis of the extraocular muscle, an autosomal dominant muscular dystrophy.