The cardiac troponin C (cTnC) mutation, L29Q, has been associated with familial hypertrophic cardiomyopathy. We observed that L29, together with neighboring residues, Asp2, Val28, and Gly30, plays an important role in determining the Ca²⁺ affinity of site II, the regulatory site of mammalian cTnC (McTnC). Here we report on the Ca²⁺ binding characteristics of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD) utilizing a F27W substitution, allowing one to monitor Ca²⁺ binding and release. We also studied the effect of these mutants on the Ca²⁺ activation of force generation in single mouse cardiac myocytes using cTnC replacement, together with the sarcomere length (SL) dependence. The Ca²⁺-binding affinity of site II of L29Q McTnC^F27W and NIQD McTnCF27W were ~1.3- and ~1.9-fold higher, respectively, than that of McTnC^F27W. The Ca²⁺-disassociation rate from site II of L29Q McTnC^F27W and NIQD McTnC^F27W were not significantly different than that of control (McTnC^F27W). However, the rate of Ca²⁺ binding to site II was higher in L29Q McTnC^F27W and NIQD McTnC^F27W relative to control (~1.5-fold and ~2.0-fold respectively). The Ca²⁺ sensitivity of force generation was significantly higher in myocytes reconstituted with L29Q McTnC (~1.4-fold) and NIQD McTnC (~2-fold). Interestingly, the change in Ca²⁺ sensitivity of force generation in response to SL change (1.9, 2.1 and 2.3 µm) was significantly reduced in myocytes containing either L29Q McTnC or NIQD McTnC. Together, these results demonstrate that the L29Q mutation enhances the Ca²⁺-binding characteristics of cTnC, and that when incorporated into cardiac myocytes, this mutant alters myocyte contractility.