Background - During ischemia, the intracellular calcium and inorganic phosphate (Pi) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the myocardial response to Ca2+, Pi, and pH. Methods and Results - Isometric force was studied in mechanically isolated Triton-skinned single myocytes from left ventricular myocardium. Force declined with added Pi to 0.33±0.02 of the control force (pH 7.1, 0 mmol/L Pi) at 30 mmol/L Pi and increased with pH from 0.64±0.03 at pH 6.2 to 1.27±0.02 at pH 7.4. Force dependency on Pi and pH did not differ between donor and failing hearts. Incubation of myocytes in a Pi-containing activating solution caused a potentiation of force, which was larger at submaximal than at maximal [Ca2+]. Ca2+ sensitivity of force was similar in donor hearts and hearts with moderate cardiac disease, but in end-stage failing myocardium it was significantly increased. The degree of myosin light chain 2 phosphorylation was significantly decreased in end-stage failing compared with donor myocardium, resulting in an inverse correlation between Ca2+ responsiveness of force and myosin light chain 2 phosphorylation. Conclusions - Our results indicate that contractile protein alterations in human end-stage heart failure alter Ca2+ responsiveness of force but do not affect the force-generating capacity of the cross-bridges or its Pi and pH dependence. In end-stage failing myocardium, the reduction in force by changes in pH and [Pi] at submaximal [Ca2+] may even be less than in donor hearts because of the increased Ca2+ responsiveness.