Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ pump is specifically phosphorylated at Ser¹⁶ and Thr¹⁷ by cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of crdiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR-CaMK incorporated the same amount of³²P into PLB-24, 0.60±0.01 nmol³²P/mg SR protein and 0.61±0.03 nmol³²P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC₅₀=0.2μM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC₅₀=1.6μM) and a CaMKII-specific inhibitor peptide (IC₅₀=2.5μM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases³²P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinasein vitro even when the adjacent site is phosphorylated.