The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male Sprague-Dawley rats at 900°/s throughout a 90°-arc of motion. Maximal tetanic tension (P_o) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (α-actinin, desmin, dystrophin, β-spectrin), integral membrane (α- and β-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P₀ was significantly reduced to 4.23 ± 0.22 N, compared with 8.24 ± 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 ± 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (β-spectrin) or not at all (α- and β-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction.