The aims of this study were to investigate the sarcomeric accumulation and expression of heat shock proteins (HSPs) after two bouts of maximal eccentric exercise. Twenty four subjects performed two bouts of 70 maximal voluntary eccentric actions using the elbow flexors in one arm. The bouts were separated by three weeks. The changes in concentric (60°^.s^-1) and isometric (90°) force-generating capacity were monitored for nine days after each bout, and biopsies were taken one and 48 hours and four and seven days after bout 1 and one and 48 hours after bout 2. The content of HSP27, B-crystallin, HSP70, and desmin in the cytosolic and cytoskeleton/myofibrilar fraction of homogenized muscle samples was determined by immunoassays, and the cellular and subcellular localization of the HSPs in the myofibrilar structure was analyzed by conventional and confocal immunofluorescence microscopy and quantitative electron microscopy. The force-generating capacity was reduced by ~50% and did not recover completely during the three weeks following bout 1. After bout 2, the subjects recovered within one week. The HSP levels increased in the cytosolic fraction after bout 1, especially HSP70 (~300% 2-7 days after exercise). Increased levels of HSP27, B-crystallin, and HSP70 were found in the cytoskeletal/myofibrilar fraction after both bouts - despite reduced damage after bout 2. At the ultrastructural level, HSP27 and B-crystallin accumulated in Z-disks, in intermediate desmin-like structures (B-crystallin), and in areas of myofibrilar disruption. In conclusion, HSP27 and B-crystallin accumulated in myofibrilar structures, especially in the Z-disks and the intermediate structures (desmin). The function of the small HSPs is possibly to stabilize and protect the myofibrilar structures during and after unaccustomed eccentric exercise. The large amount of HSP27, B-crystallin, and HSP70 in the cytoskeletal/myofibrilar fraction after a repeated bout of exercise suggests a protective role as part of the repeated-bout effect.