It is well known that cardiac troponin C possess only one regulatory Ca²⁺-binding site while fast skeletal troponin C possess two of them. However, the consequence of the second Ca²⁺-binding site for the kinetic mechanism by which troponin switches on muscle contraction remains unknown. We have previously shown that a simple two step mechanism can account for the full [Ca²⁺]-dependence of the biphasic fluorescence changes observed upon rapidly mixing Ca²⁺ at different concentrations with cardiac myofibrils having incorporated site-specifically labelled cardiac troponin. Applying the same experimental technique to skeletal troponin incorporated into myofibrils from fast skeletal muscle reveals biphasic kinetics as well, with rate constants increasing from 250 s^-1 at low [Ca²⁺] to 1000 s^-1 at high [Ca²⁺] for the fast phase and from 15 s^-1 to 150 s^-1 for the slow phase. However, in contrast to the fluorescence changes observed for cardiac troponin, it is not possible to simulate the [Ca²⁺]-dependence of the ratio of the amplitudes for the two phases by a simple model consisting of two sequential conformational changes. Instead, successful simulation of the fluorescence transients observed with skeletal troponin requires to separate the two conformational changes producing the fluorescence changes by an interjacent, additional Ca²⁺-binding step. A thermodynamic consistent model is proposed in which the binding of the first Ca²⁺ to skeletal troponin C leads to a fast conformational change of troponin that facilitates the binding of the second Ca²⁺. The latter is required to enable the observed slower conformational change that regulates skeletal muscle contraction.