Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, functioning to modulate contractile force by altering the rate of calcium re-sequestration by the Ca-ATPase. Functionally, inhibition by PLB binding is manifested by shifts in the calcium dependence of Ca-ATPase activation toward higher calcium levels; phosphorylation of PLB by PKA reverses the inhibitory action of PLB. To investigate structural changes in the cytoplasmic portion of PLB that result from either the phosphorylation of PLB by cAMP-dependent protein kinase (PKA) or calcium binding to the Ca-ATPase, we have used frequency-domain fluorescence spectroscopy to measure the spatial separation and conformational heterogeneity between N-(1-pyrenyl)maleimide, covalently bound to a single cysteine (Cys(24)) engineered near the membrane surface of the transmembrane domain of PLB, and Tyr(6) in the cytosolic domain. Irrespective of calcium activation of the Ca-ATPase or phosphorylation of Ser(16) in PLB by PKA, we find that PLB remains tightly associated with the Ca-ATPase in a well-defined conformation. However, calcium activation of the Ca-ATPase induces an increase in the overall dimensions of the cytoplasmic portion of bound PLB, whereas PLB phosphorylation results in a more compact structure, consistent with increased helical content induced by a salt link between phospho-Ser(16) and Arg(13). Thus, enzyme activation of the Ca-ATPase may occur through different mechanisms: calcium binding to high-affinity sites within the Ca-ATPase functions to overcome conformational constraints imposed by PLB on the N-domain of the Ca-ATPase; alternatively, phosphorylation stabilizes the backbone fold of PLB to release inhibitory interactions with the Ca-ATPase.