Synemin is an intermediate filament protein shown previously to interact with alpha-dystrobrevin and desmin. Immunoblot analysis detects a beta-synemin protein of 170 kDa in human skeletal muscle and an alpha-synemin protein of 225 kDa in monkey brain. Low-resolution immunohistochemical analysis localizes beta-synemin within muscle along the sarcolemma, whereas confocal microscopic analysis further refines localization to the costamere and muscle Z-lines. In addition to these locations, beta-synemin is also enriched at the neuromuscular and myotendinous junctions, other regions that undergo high stress during myofiber contraction. Based on its localization and its expression pattern, it is proposed that beta-synemin functions as a structural protein involved in maintaining muscle integrity through its interactions with alpha-dystrobrevin, desmin, and other structural proteins.