The positive force-frequency relation, one of the key factors modulating performance of healthy myocardium, has been attributed to an increased Ca2+ influx per unit of time. In failing hearts, a blunted, flat or negative force-frequency relation has been found. In healthy and failing hearts frequency-dependent alterations in Ca2+ sensitivity of the myofilaments, related to different phosphorylation levels of contractile proteins, could contribute to this process. Therefore, the frequency dependency of force, intracellular free Ca2+ ([Ca2+]i), Ca2+ sensitivity and contractile protein phosphorylation were determined in control and monocrotaline-treated, failing rat hearts. An increase in frequency from 0.5 to 6 Hz resulted in an increase in force in control (14.3 ± 3.0 mN mm-2) and a decrease in force in failing trabeculae (9.4 ± 3.2 mN mm-2), whereas in both groups the amplitude of [Ca2+]i transient increased. In permeabilized cardiomyocytes, isolated from control hearts paced at 0 and 9 Hz, Ca2+ sensitivity remained constant with frequency (pCa50: 5.55 ± 0.02 and 5.58 ± 0.01, respectively, P > 0.05), whereas in cardiomyocytes from failing hearts Ca2+ sensitivity decreased with frequency (pCa 50: 5.62 ± 0.01 and 5.57 ± 0.01, respectively, P < 0.05). After incubation of the cardiomyocytes with protein kinase A (PKA) this frequency dependency of Ca2+ sensitivity was abolished. Troponin I (TnI) and myosin light chain 2 (MLC2) phosphorylation remained constant in control hearts but both increased with frequency in failing hearts. In conclusion, in heart failure frequency-dependent myofilament Ca2+ desensitization, through increased TnI phosphorylation, contributes to the negative force-frequency relation and is counteracted by a frequency-dependent MLC2 phosphorylation. We propose a novel role for PKC-mediated TnI phosphorylation in modulating the force-frequency relation.