The N-terminal extension of human cardiac troponin T (TnT), which modulates myofilament Ca sensitivity, contains several hypertrophic cardiomyopathy (HCM)-causing mutations including S69F. However, the functional consequence of S69F mutation is unknown. The human analog of S69F in rat TnT is L71F (TnT). Because the functional consequences due to structural changes in the N-terminal extension are influenced by the type of myosin heavy chain (MHC) isoform, we hypothesized that the TnT-mediated effect would be differently modulated by α- and β-MHC isoforms. TnT and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (β-MHC) to measure steady-state and dynamic contractile parameters. The magnitude of the TnT-mediated attenuation of Ca-activated maximal tension was greater in α- than in β-MHC fibers. For example, TnT attenuated maximal tension by 31% in α-MHC fibers but only by 10% in β-MHC fibers. Furthermore, TnT reduced myofilament Ca sensitivity by 0.11 pCa units in α-MHC fibers but only by 0.05 pCa units in β-MHC fibers. TnT augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in β-MHC fibers. Collectively, our data demonstrate that TnT induces greater contractile deficits against α-MHC than against β-MHC background.