We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnCF27W: xxsTnC. It is interesting that pCa50 increased by approximately 0.1 for M80Q sTnC and approximately 0.3 for M80Q sTnCF27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, nH decreased to a similar value for all mutant sTnCs. Altered sTnC Ca2+-binding properties (M80Q sTnCF27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca2+ affinity strongly influences Ca2+ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca2+ to thin filament activation.