Protein kinase A (PKA)-dependent phosphorylation of sarcomeric proteins, which is believed to contribute significantly to the regulation of cardiac contraction, has been studied extensively in skinned myocardium. However, whereas most reports focused on effects of phosphorylation, little is known about reverse effects of dephosphorylation. To analyze the impact of dephosphorylation of PKA sites on troponin I (TnI), the endogenous troponin of murine triton-skinned cardiac fibers was exchanged with troponin containing the phospho-mimicking mutant TnIS22/23D , remaining PKA substrates were phosphorylated by incubation with recombinant PKA, and effects of subsequent dephosphorylation on the Ca2+ -sensitivity of contraction were examined using the catalytic subunit of protein phosphatase 1 (PP1c) in the presence of Mn2+ . In fibers exchanged with TnIS22/23D PKA incorporated 32 P only into myosin binding protein C whereas wildtype TnI was phosphorylated significantly. Sequential incubation of PKA pre-treated fibers containing TnIS22/23D with Mn2+ -containing buffer and PP1c increased the Ca2+ -sensitivity by 0.11 pCa-units whereas in fibers containing wildtype TnI the Ca2+ -sensitivity increased by 0.20 pCa-units. In conclusion these data indicate that Ser-22 and Ser-23 of TnI play an important role in mediating the shifts in Ca2+ -sensitivity, however, dephosphorylation of other substrates may contribute.