Objective: The alterations in contractile proteins underlying enhanced Ca²⁺-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca²⁺-responsiveness in human heart failure. Methods: Isometric force and its Ca²⁺-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca²⁺-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. Results: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca²⁺-sensitivity of the contractile apparatus (pCa₅₀) was significantly higher in failing myocardium (ΔpCa₅₀=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca²⁺-responsiveness. An inverse correlation was found between pCa₅₀ and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca²⁺-sensitivity to a larger extent in failing (ΔpCa₅₀=0.20) than in donor (ΔpCa₅₀=0.03) myocytes, abolishing the difference in Ca²⁺-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca²⁺-responsiveness. Conclusions: The increased Ca²⁺-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.