This spectroscopic study examines the steady state and kinetic parameters governing the crossbridge effect necessary to increase the Ca2+ affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants to the level seen in skinned fibers. Previously, it was shown by Landstrom, et al. (J. Mol. Cell Card. 45:281-288; 2008) and Pinto, et. al. (J. Biol. Chem 284(28): 19090-19100; 2009) that the cTnC mutations A8V, C84Y, E134D and D145E do not increase the apparent Ca2+ affinity of isolated cTnC (D145E shows a slight increase) as monitored by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid (IAANS) fluorescence. Follow-up experiments showed that when cTnC mutants are incorporated into regulated thin filaments (RTF), only the A8V mutant increased the apparent Ca2+ affinity. Addition of myosin subfragment-1 (S1) to mutant RTFs (in the absence of ATP) increased the apparent Ca2+ affinity to similar levels seen in cTnC mutant reconstituted skinned fibers. Therefore, strong crossbridges were required to fully alter the apparent cTnC Ca2+ affinity and recapitulate the changes observed in the Ca2+ sensitivity of tension. Stopped flow fluorescence techniques were also used to measure the kinetics of Ca2+ binding to troponin complex (cTn) and RTF prepared with IAANS labeled cTnC mutants. At the cTn level, both A8V and D145E cTnC decreased the rate of Ca2+ dissociation; while in the RTF, only A8V decreased the rate of Ca2+ dissociation. Future experiments will determine the rate of Ca2+ dissociation from RTFs in the presence of S1. This study indicates that although these HCM-cTnC mutants display similar phenotypes in skinned fibers, they utilize different molecular mechanisms to alter the Ca2+-sensitivity of skinned muscle. Supported by NIH HL-42325 (JDP) and AHA 0825368E (JRP) and AHA 09POST2300030 (MSP).