Cardiac troponin T (cTnT) has a highly acidic extended N-terminus, the physiological role of which remains poorly understood. To decipher the physiological role of this unique region, we deleted specific regions within the N-terminus of mouse cTnT (McTnT) to create McTnT(1-44) and McTnT(45-74) proteins. Contractile function and dynamic force-length measurements were made after reconstituting the McTnT deletion proteins into detergent-skinned cardiac papillary fibres harvested from nontransgenic mice that expressed α-tropomyosin (Tm). To further understand how the functional effects of N-terminus of cTnT are modulated by Tm isoforms, McTnT deletion proteins were reconstituted into detergent-skinned cardiac papillary fibres harvested from transgenic mice that expressed both α- and β-Tm. McTnT(1-44), but not McTnT(45-74), attenuated maximal activation of the thin filament. Myofilament Ca(2+) sensitivity, as measured by pCa(50) (-log of [Ca(2+)](free) required for half maximal activation), decreased in McTnT(1-44) (α-Tm) fibres. The desensitizing effect of McTnT(1-44) on pCa(50) was ablated in β-Tm fibres. McTnT(45-74) enhanced pCa(50) in both α- and β-Tm fibres, with β-Tm having a bigger effect. The Hill coefficient of tension development was significantly attenuated by McTnT(45-74), suggesting an effect on thin filament cooperativity. The rate of crossbridge (XB) detachment and the strained XB-mediated impact on other XBs were augmented by McTnT(1-44) in β-Tm fibres. The magnitude of the length-mediated recruitment of XBs was attenuated by McTnT(1-44) in β-Tm fibres. Our data demonstrates that the 1-44 region of McTnT is essential for maximal activation, whereas the cardiac-specific 45-74 region of McTnT is essential for augmenting cooperativity. Moreover, our data shows that α- and β-Tm isoforms have divergent effects on McTnT deletion mutant's ability to modulate cardiac thin filament activation and Ca(2+) sensitivity. Our results not only provide the first explicit evidence for the existence of two distinct functional regions within the N-terminus of cTnT, but also offer mechanistic insights into the divergent physiological roles of these regions in mediating cardiac contractile activation.