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 6Hz resulted in an increase in force in control (14.3±3.0 mN/mm²) and a decrease in force in failing trabeculae (9.4±3.2 mN/mm²), whereas in both groups the amplitude of [Ca²⁺]_i transient increased. In permeabilised cardiomyocytes, isolated from control hearts paced at 0 and 9Hz, 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 (pCa50: 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⁺ desensitisation, 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 relationship.