Phospholamban is a small integral membrane protein of cardiac, smooth, and slow‐twitch skeletal muscle sarcoplasmic reticulum that interacts with the Ca²⁺ pump of these organelles and inhibits Ca²⁺‐pump activity while in the dephosphorylated form. Three sites of Ser/Thr phosphorylation have been identified in the primary sequence of phospholamban, at Ser‐10, Ser‐16, and Thr‐17. In vitro studies indicate that these residues are phosphorylated by PKC (Ser‐10), PKA, PKG or PKC (Ser‐16), and CaM kinase II (Thr‐17). Phosphorylation of Ser‐16 (or Thr‐17) is accompanied by an increase in Ca²⁺ pump activity in direct proportion to the stoichiometry of phosphorylation. Dual phosphorylation of both Ser‐16 and Thr‐17 does not cause any further stimulation of pump function over that achieved by stoichiometric phosphorylation of a single site. Examination of the pattern of phosphorylation in vivo has been aided by the generation of polyclonal antibodies specific for the phosphorylated forms of phospholamban. β‐Adrenergic stimulation of cardiac muscle results in phosphorylation of both Ser‐16 and Thr‐17. The time course of Ser‐16 phosphorylation precedes Thr‐17. The spatial distribution of Ser‐16 and Thr‐17 phosphorylated forms of phospholamban is not identical; phospholamban located in the nuclear membrane of a cardiac myocyte is phosphorylated exclusively on Ser‐16, whereas phospholamban molecules in the SR membrane of the same cell are phosphorylated on Ser‐16 and/or Thr‐17. Finally, we have identified a novel stimulus for the phosphorylation of phospholamban. Ca²⁺ store depletion, achieved by exposure of myocytes to SERCA inhibitors, prompts the phosphorylation of phospholamban on Ser‐16. This would be expected to increase Ca²⁺ uptake by the SR in an attempt to achieve the refilling of the SR.