The objective of this study was to evaluate the effect of steak location and postmortem aging on cooked meat tenderness and myofibrillar protein degradation of steaks from M. semitendinosus (ST). Following harvest and a 6 d chill period, the left ST was removed from carcasses of crossbred feedlot steers ( = 60, average hot carcass weight 427 ± 24 kg). Each ST was fabricated into ten 2.54-cm thick steaks originating from the proximal to distal end of the muscle. Steaks cut adjacent to each other were paired, vacuum packaged, and randomly assigned to 7, 14, 21, 42, or 70 d of aging at 2 ± 1°C. After aging, within each steak pair, steaks were randomly assigned to Warner-Bratzler shear force or myofibrillar proteolysis analysis (calpain activity and desmin and troponin-T degradation). Muscle fiber type and size were also determined at the 2 ends of the muscle. There was no location × d of aging interaction ( = 0.25) for ST steak WBSF. Steak location affected (quadratic, < 0.01) WBSF. As steaks were fabricated from the proximal to distal end, WBSF values decreased toward the middle of the muscle and then increased toward the distal end. Activity of all calpains and myofibrillar protein proteolysis were unaffected by steak location ( > 0.13). Type I, IIA, and IIX muscle fibers were larger at the proximal end of the muscle than the distal end ( < 0.01). Increasing d of aging improved WBSF (quadratic, < 0.01) for the duration of the 70 d postmortem period. As d of aging increased, intact calpain-1 activity decreased (quadratic, < 0.01) with activity detected through 42 d. Day of aging affected autolyzed calpain-1 (linear, < 0.01) and calpain-2 activity (quadratic, < 0.01). Through d 70 of aging, the intensity of intact 55 kDa desmin band decreased (linear, < 0.01), while there was an increase (linear, < 0.01) in the degraded 38 kDa band. Similarly, d of aging increased troponin-T proteolysis, indicated by a decrease (quadratic, < 0.01) in intensity of the intact 40 kDa band and an increase (linear, < 0.01) in the 30 kDa degraded band. Intramuscular WBSF differences are not due to proteolytic activity or myofibrillar degradation and seem related to muscle fiber size. The improvement of ST steak WBSF through 70 d of aging is partly due to continued degradation of desmin and troponin-T. Calpain proteolytic analysis indicates that autolyzed calpain-1 and calpain-2 may be involved in extended postmortem myofibrillar protein proteolysis.