The phosphorylation status of myofibrillar proteins influences the Ca2+ responsiveness of the myofilaments,but the contribution of and the interaction between the individual components is poorly characterized. Therefore, in Langendorff perfused rat hearts (n=30), the phosphorylation levels of cardiac myosin binding protein-C (cMyBP-C), troponin I and T (cTnI, cTnT) and myosin light chain 1 and 2 (MLC-1, MLC-2) were determined by 1- and 2-dimensional gel electrophoresis. Isometric force development, its Ca2+-sensitivity, the rate of tension redevelopment (ktr) and passive force (Fpas) were studied at optimal sarcomere length (2.2 μm) in mechanically isolated,permeabilized cardiomyocytes at 15 7deg;C. Protein phosphorylation was varied by: 1) blocking spontaneous cardiac activity by lidocaine (0.35 mM; Quiescence); 2) electrical stimulation of the hearts at 5 Hz (Contraction) and 3. treatment of contracting hearts with Isoprenaline (1 μM). MLC-2 phosphorylation was increased in the Contraction group almost 2-fold, relative to the Quiescence group, whereas cMyBP-C and cTnI phosphorylation remained the same. Isoprenaline resulted in 3.7-fold increases in both cMyBP-C and cTnI phosphorylation, but did not result in a further increase in MLC-2 phosphorylation.No significant differences were found in maximum force and ktr between groups, both before and after protein kinase A (PKA) treatment. Ca2+-sensitivity in the Contraction and Isoprenaline groups was significantly reduced in comparison to the Quiescence group. These differences were largely abolished by PKA and Fpas was reduced. These results highlight the impact of PKA-dependent phosphorylation on Ca2+-sensitivity and provide evidence for an interaction between the effects of TnI and MLC-2 phosphorylation.