We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium‐binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt‐Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine–glutamine exchange at position 29 in the nonfunctional calcium‐binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N‐terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107–112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A‐dependent phosphorylation of cTnI at serine 22 and serine 23 in wild‐type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+‐free and Ca2+‐saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+‐dependent actomyosin subfragment 1 (actoS1)‐ATPase activity and the sliding velocity of thin filaments. The maximum actoS1‐ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A‐dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1‐ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A‐dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.