Dilated cardiomyopathy and hypertrophic cardiomyopathy (HCM) can be caused by mutations in thin filament regulatory proteins of the contractile apparatus. In vitro functional assays show that, in general, the presence of dilated cardiomyopathy mutations decreases the Ca²⁺ sensitivity of contractility, whereas HCM mutations increase it. To assess whether this functional phenomenon was a direct result of altered Ca²⁺ affinity or was caused by altered troponin–tropomyosin switching, we assessed Ca²⁺ binding of the regulatory site of cardiac troponin C in wild-type or mutant troponin complex and thin filaments using a fluorescent probe (2-[4′-{iodoacetamido}aniline]-naphthalene-6-sulfonate) attached to Cys35 of cardiac troponin C. The Ca²⁺-binding affinity (pCa₅₀=6.57±0.03) of reconstituted troponin complex was unaffected by all of the HCM and dilated cardiomyopathy troponin mutants tested, with the exception of the troponin I Arg145Gly HCM mutation, which caused an increase (ΔpCa₅₀=+0.31±0.05). However, when incorporated into regulated thin filaments, all but 1 of the 10 troponin and α-tropomyosin mutants altered Ca²⁺-binding affinity. Both HCM mutations increased Ca²⁺ affinity (ΔpCa₅₀=+0.41±0.02 and +0.51±0.01), whereas the dilated cardiomyopathy mutations decreased affinity (ΔpCa₅₀=−0.12±0.04 to −0.54±0.04), which correlates with our previous functional in vitro assays. The exception was the troponin T Asp270Asn mutant, which caused a significant decrease in cooperativity. Because troponin is the major Ca²⁺ buffer in the cardiomyocyte sarcoplasm, we suggest that Ca²⁺ affinity changes caused by cardiomyopathy mutant proteins may directly affect the Ca²⁺ transient and hence Ca²⁺-sensitive disease state remodeling pathways in vivo. This represents a novel mechanism for this class of mutation.