In cardiac myocytes, the activity of the Ca²⁺/calmodulin-dependent kinase II (CaMKII) is hypothesized to regulate Ca²⁺ release from and Ca²⁺ uptake into the sarcoplasmic reticulum via phosphorylation of the ryanodine receptor and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of [Ca²⁺]_i-transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies ranging from 0.2 to 10 Hz and at 37 ^oC). Compared to mice lacking PLN only, combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca²⁺ release rate by more than 50% at 10 Hz. While PLN ablation increased the rate of Ca²⁺ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca²⁺]_i-transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca²⁺]_i-transient, indicating that PLN-mediated feedback on [Ca²⁺]_i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes (1) combined chronic CaMKII inhibition and PLN ablation slowed Ca²⁺ release at physiological frequencies, (2) frequency-dependent decay of the amplitude and shortening of the [Ca²⁺]_i-transient occurs independent of chronic CaMKII inhibition and PLN ablation, (3) PLN-mediated regulation of Ca²⁺ uptake is diminished at higher stimulation frequencies within the physiological range.