Objective: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1±2.0%) compared to donor (31.9±4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. Methods: Cooperative effects in isometric force and rate of force redevelopment (Ktr) were studied in single Triton-skinned human cardiomyocytes at various [Ca2+] before and after protein phosphatase-1 (PP-1) incubation. Results: Maximum force and Ktr values did not differ between failing and donor hearts, but Ca2+-sensitivity of force (pCa50) was significantly higher in failing myocardium (ΔpCa50=0.17). Ktr decreased with decreasing [Ca2+], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa50 to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute Ktr values was found after PP-1 in failing and donor myocytes, while the shape of the Ktr-Ca2+ relationships remained unaltered. Conclusions: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca2+-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca2+-sensitivity and impaired Ca2+-handling on diastolic function in human heart failure.