The human skeletal muscle yeast two-hybrid cDNA library was screened with the carboxyl-terminal region (the last 200 amino acids) of dystrophin. Two interacting clones were identified corresponding to alpha-actinin-2 and actin. Interactions between alpha-actinin, actin, and dystrophin were confirmed by the ligand-blotting technique, by colocalization of dystrophin and alpha-actinin-2 to the isolated skeletal muscle sarcolemmal vesicles and to the plasma membranes isolated from C2C12 myoblasts, and by indirect immunolocalization of dystrophin and alpha-actinin-2 in skeletal muscle cells. This is the first identification of a direct interaction between alpha-actinin, actin, and the carboxyl-terminal region of dystrophin. We propose that dystrophin forms lateral, multicontact association with actin and that binding of alpha-actinin-2 to the carboxyl-terminus of dystrophin is the communication link between the integrins and the dystrophin/dystrophin-glycoprotein complex.