Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.