The organization of the dystrophin glycoprotein complex (DGC) was studied by investigating interactions between its components. For this purpose, mouse dystrophin and syntrophin-1 (alpha-syntrophin) sequences were expressed as chimeric fusion proteins and used in overlay binding experiments to probe gel blots of purified rabbit muscle DGC. In order to identify the DGC proteins that bind to different regions of dystrophin, the amino-terminal 385 amino acids, the unique carboxy-terminal domain (amino acids 3266-3678), and the adjacent cysteine-rich region of dystrophin homologous to alpha-actinin (amino acids 3074-3265) were expressed as separate fusion proteins. The cysteine-rich sequences of dystrophin predominantly bound adhalin (gp50) and to full length dystrophin suggesting that these sequences may also be important to dystrophin dimerization. The carboxy-terminal domain sequences strongly bound all of the DGC syntrophins and weakly, adhalin, while the amino-terminal sequences of dystrophin bound none of the proteins of this complex. Fusion proteins containing alpha-syntrophin sequences bound not only to dystrophin but also to all three DGC syntrophins, adhalin, and gp35. The interactions identified here were used to refine the existing model of DGC organization to make it consistent with the current data.