Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca 2+ -sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca 2+ -sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66±1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca 2+ -sensitivity (pCa 50 ) to 5.50±0.02 relative to the donor control value (pCa 50 =5.43±0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca 2+ -sensitivity relative to the failing control (pCa 50 =5.60±0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca 2+ -sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono-and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue.These differences in Ca 2+ -sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca 2+ -sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca 2+ -sensitivity.