Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (α, β, γ, δ). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of β-, γ- and δ-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the β/δ-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.