Troponin T (TnT) is an essential component of troponin (Tn) for the Ca²⁺-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH₂-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal β-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca²⁺ binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 Å, respectively in the absence of Ca²⁺, and increased by 2.6 and 5.8 Å, respectively upon binding of Ca²⁺ to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 Å respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca²⁺- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca²⁺, the rigor S1 binding also increased the distances to the same extent as the presence of Ca²⁺ when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca²⁺. However, plots of the extent of S1-incuced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca²⁺ but sigmoidal curve in the absence of Ca²⁺. FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca²⁺-induced or closed, and Sl-induced or open states).