In human failing myocardium, an increased Ca2+-sensitivity of myofilament tension development has been described in Triton X skinned cardiac myocytes compared to cardiomyocytes obtained from non-failing human donor hearts. The present study aimed to investigate whether there are functional implications of the increased Ca2+-sensitivity in heart failure and whether alterations of myofilament function are already obvious at earlier stages of heart failure, such as in cardiac hypertrophy or whether alterations of the intracellular Ca2+-homeostasis are able to induce alterations in myofilament function. Ca2+-activated tension development was measured in Triton X-skinned fibers from human failing and non-failing myocardium. Ca2+-sensitivity of myofilament tension development was significantly shifted to the left in human failing myocardium. Plots of diastolic free Ca2+ versus diastolic tension development showed that in a range of similar diastolic Ca2+-concentrations, diastolic tension was significantly enhanced in the failing hearts. The Ca2+/tension relationship was shifted to the right in Triton X-skinned fiber preparations from transgenic renin overexpressing rats (TG(mREN2)27), shown to have concentric hypertrophy. In addition, the Ca2+/tension relationship was unchanged in phospholamban knock-out mice with an increased systolic Ca2+ (and enhanced diastolic Ca2+-load). It is concluded that the increased Ca2+-sensitivity of myofilament tension observed in single cardiomyocytes from failing human myocardium may be a phenomenon also present in multicellular preparations and may contribute to the diastolic dysfunction observed in human heart failure. Alterations of myofilament function occur at very early stages of heart failure and may be species dependent, or dependent on intracellular free Ca2+-levels.