Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at <10 nM free Ca2+. 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca2+, even without high-affinity cross-bridge binding. They also shifted the force-pCa curve of muscle fibers to lower Ca2+ by 0.8-1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force-pCa were maintained in the presence of para-aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.