Although it has been suggested that in cardiac muscle the phosphorylation level of myosin regulatory light chain (RLC) correlates with frequency of stimulation, its significance in the modulation of the force–frequency and pressure–frequency relationships remains unclear. We examined the role of RLC phosphorylation on the force–frequency relation (papillary muscles), the pressure–frequency relation (Langendorff perfused hearts) and shortening–frequency relation (isolated cardiac myocytes) in nontransgenic (NTG) and transgenic mouse hearts expressing a nonphosphorylatable RLC protein (RLC(P−)). At 22 °C, NTG and RLC(P−) muscles showed a negative force–frequency relation. At 32 °C, at frequencies above 1 Hz, both groups showed a flat force–frequency relation. There was a small increase in RLC phosphorylation in NTG muscles when the frequency of stimulation was increased from 0.2 Hz to 4.0 Hz. However, the level of RLC phosphorylation in these isolated muscles was significantly lower compared to samples taken from NTG intact hearts. In perfused hearts, there was no difference in the slope of pressure–frequency relationship between groups, but the RLC(P−) group consistently developed a reduced systolic pressure and demonstrated a decreased contractility. There was no difference in the level of RLC phosphorylation in hearts paced at 300 and 600 bpm. In RLC(P−) hearts, the level of TnI phosphorylation was reduced compared to NTG. There was no change in the expression of PLB between groups, but expression of SERCA2 was increased in hearts from RLC(P−) compared to NTG. In isolated cardiac myocytes, there was no change in shortening–frequency relationship between groups. Moreover, there was no change in Ca²⁺ transient parameters in cells from NTG and RLC(P−) hearts. Our data demonstrate that in cardiac muscle RLC phosphorylation is not an essential determinant of force– and pressure–frequency relations but the absence of RLC phosphorylation decreases contractility in force/pressure developing preparations.