The C-domain Ca-Mg binding sites III and IV of troponin C (TnC) control TnC-thin filament (T.F.) affinity but are difficult to study in fibers because they do not cause tension. Here we measured the physiological K_0.5 (apparent affinity)^-1 for the binding of Ca²⁺ to the C-terminal domain of TnC in the presence or absence of rigor bonds without Mg²⁺. Ca²⁺ binding was estimated from TnC efflux from the T.F. in the presence of different [Ca²⁺], using skinned skeletal psoas fibers reconstituted with chicken recombinant (r) or native (s) TnC's that are known from studies in solution to differ in C-terminal Ca²⁺ affinity (pK_0.5 6.7 vs 7.1) due to substitution of Ile for Thr130. We also measured pK_0.5 for IT130, a recombinant TnC that has Thr130 and the same pK_0.5 in solution as sTnC. TnC loss due to exposure to Rigor+Ca²⁺ or R+Ca²⁺ was monitored by testing isometric tensions under standard conditions (pCa 4.4, pMgATP 2.3, Kpropionate 152mM, pH 7, 15º C). In the relaxed state (R, with 0.1mM MgATP and 1mM free Mg²⁺) pK_0.5 values of rTnC, sTnC and IT130 were pCa 8.4-8.6, while in rigor condition the pK_0.5 values were pCa 9.0-9.2 . These results show that Ca²⁺ affinity for the C-terminal domain of TnC in its biological environment increases ~ 100x compared to the measurements done in solution; that the difference of 0.4 pCa units between rTnC and sTnC in solution is lost in the in situ experiments; and that the formation of rigor crossbridges enhances  the C-domain Ca²⁺ binding about five fold. The next step will be to perform these experiments in the presence of a physiological concentrations of  Mg²⁺( 2 mM ).