We developed a novel technique to study the Ca²⁺-induced switch kinetics of the regulatory troponin complex (Tn) in its native structural environment of the contractile sarcomere. Tn was isolated from rabbit muscle and labelled with fluorescein almost exclusively binding to a single site (Cys133 in the inhibitory subunit, TnI). The labelled Tn was exchanged against endogenous Tn in rabbit psoas myofibrils. These myofibrils were put under relaxing [Ca²⁺] in a stopped-flow apparatus to be mixed with different activating [Ca²⁺], recording the kinetics of the Tn-switch from its off-state to its on-state. Traces exhibited an initial lag phase followed by a monoexponential rise in fluorescence. The rate constants of both, the lag and rise increased with elevating the activating [Ca²⁺] to reach maxima of ~300 ± 20 s^-1 and ~200 ± 20 s^-1 at fully activating [Ca²⁺], respectively. For all Ca²⁺-activation levels the Tn-switch kinetics were considerably faster than the mechanically measured kinetics of Ca²⁺-induced myofibrillar force development which yields rate constants of ~6 ± 1 s^-1 at saturating [Ca²⁺]. This indicates that thin filament activation does not rate limit force development.