The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins α-actinin and plectin and membrane scaffolding proteins dystrophin and β-spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and αB-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and αB-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and αB-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site.