We have engineered the Ca2+ binding properties of troponin C (TnC) to study the role of increased (I60Q sTnC) and decreased (M80Q sTnCF27W) Ca2+ dissociation rate (koff) on activation and relaxation of skeletal muscle. Previously we reported that myofibril force development kinetics (kACT) are not influenced by decreasing koff from Tn, but are slowed by an increase in koff (Kreutziger et al. 2008 JPhsiol. 586;3683-3700) at low [Pi] (5 μm). The time to initiation of force (kAlag) following a rapid (∼10ms) switch from pCa 9.0 to pCa 3.5 provides information about thin filament activation rate and our preliminary data suggest this rate may also be sensitive to koff. In rabbit psoas myofibrils (15°C) kAlag (∼20 ms for native or WT sTnC) is almost eliminated for M80Q sTnCF27W and increased by I60Q sTnC (∼40-50 ms). Additionally, though kACT is similar for force increases from either full or partial activation to full activation, kAlag disappears when starting from partial activation. We have also reported that fast and slow phase rates of relaxation are not affected by koff, but that duration of the slow phase is affected in skeletal myofibrils. Here we report that lag prior to initiation of the slow phase (kRlag) may be also influenced by koff. Opposite to kAlag, kRlag (∼20 ms for WT or native sTnC) was increased (∼40-50 ms) by decreased koff (M80Q sTnCF27W) and almost eliminated by increased koff (I60QsTnC). These experiments demonstrate a potential approach to study thin filament activation/deactivation kinetics without the need for fluorescent probes attached to thin filament proteins that can affect their function. Supported by Telethon GGP07133, MIUR (CP, CT), NIH-HL65497 (MR).