In cardiac muscle, stimulation of β-adrenergic receptors leads to activation of PKA via the second messenger cAMP. PKA-dependent phosphorylation of several targets subsequently amplifies the L-type Ca2+ current, relieves the SERCA inhibition by PLB and is proposed to increase the Ca2+ sensitivity of the RyRs (by phosphorylation at serine 2808). Together, these mechanisms account for positive inotropy, but can also be arrhythmogenic via facilitation of spontaneous Ca2+ waves. Unexpectedly, the level of Ca2+ inside the SR ([Ca2+]SR) needed to initiate such waves has been reported to increase upon β-adrenergic stimulation, an observation which cannot be easily reconciled with elevated Ca2+ sensitivity of the RyRs. We tested the hypothesis that this change of Ca2+ wave threshold could occur indirectly, subsequent to SERCA disinhibition. Cytosolic and intra-SR Ca2+ waves were simultaneously recorded with confocal line-scans in permeabilized mouse cardiomyocytes with rhod-2 and fluo-5-N, respectively. We analyzed changes of SR Ca2+ content and several Ca2+ wave parameters. Ochratoxin A (OTA) was used to specifically stimulate the SERCA and the findings were compared to those in the presence of cAMP, which besides SERCA stimulation also leads to RyR phosphorylation. While cAMP resulted in a modest increase of Ca2+ wave thresholds (F/F0 9%±3.5%), OTA lead to a substantial increase (30±5.1%). Both compounds resulted in an acceleration of SR refilling (OTA earlier than cAMP), confirming SERCA stimulation. While cAMP provoked in the occurrence of Ca2+ sparks and mini waves, possibly accounting for the less pronounced SR loading, this was not observed in OTA. Together these results suggest that SERCA stimulation alone can elevate the intra-SR threshold for the generation of Ca2+ waves, independently of RyR phosphorylation. Supported by SNF.