Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca2+ uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca2+ release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs.Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca2+ release in normal VMs. Ca2+ sparks and spontaneous Ca2+ waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca2+ fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca2+ sparks in VMs exposed to 50nM free [Ca2+]. At physiological diastolic free [Ca2+] (100-200nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca2+], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca2+ sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca2+ release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca2+ dynamics and rhythmic activity of VMs.