Troponin T (TnT) is an essential protein in the transduction of the Ca²⁺-binding signal that triggers striated muscle contraction. Functional diversity among various TnT isoforms found in cardiac and skeletal muscles has been correlated with the sequence heterogeneity at the amino (N-) and the carboxyl (C-) terminal regions. The most striking difference between cardiac TnT (cTnT) and skeletal TnT (sTnT) is that cTnT has an extended N-terminus, which is rich in negatively charged amino acids. To investigate the role of this region in cTnT, we deleted the first 76 amino acids in rat cTnT (cTnT_77–289) by site-directed mutagenesis. We exchanged the native troponin complex in rat cardiac myofibrillar preparations and detergent skinned cardiac fiber bundles by treatment with excess cTnT or cTnT_77–289. After reconstituting the cTnT_77–289 containing myofibrils with cardiac troponin I–cardiac troponin C (cTnI–cTnC), the MgATPase activity was 70% of the cTnT treated myofibrils in the relaxed state and 83% of the cTnT treated myofibrils in the maximal Ca²⁺-activated state. These observations were supported by force measurements in which cTnT and cTnT_77–289 were exchanged into skinned fiber bundles. Prior to reconstitution with cTnI–cTnC, the Ca²⁺-independent maximal force developed by the cTnT_77–289 containing fiber was 45% of the force developed by the cTnT containing fiber. After reconstituting with cTnI–cTnC, the Ca²⁺-activated maximal force of the cTnT_77–289 containing fiber was 62% of the force developed by the cTnT containing +cTnI–cTnC reconstituted fiber. In both assays, no significant changes in the normalized Ca²⁺-activity relation or in co-operativity were observed. Fluorescence experiments using pyrene-labeled Tm demonstrated that the binding of cTnT_77–289 to Tm was 3–4 fold stronger than that of cTnT. Our results suggest that strong interactions between cTnT_77–289 and Tm stabilize cardiac myofilaments in a sub-maximally activated state. Our findings also indicate that the N-terminus of cTnT is essential for maximal activation of cardiac myofilaments.