P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca2+-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca2+ pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates1,2. An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca2+-binding sites ready to accept new cytosolic Ca2+. In the absence of Ca2+ and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca2+)3, and if millimolar Mg2+ is present, one Mg2+ is expected to occupy one of the Ca2+-binding sites with a millimolar dissociation constant4,5. This Mg2+ accelerates the reaction cycle4, not permitting phosphorylation without Ca2+ binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg2+ state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin6, a small regulatory membrane protein of Ca2+-ATPase7, is bound, stabilizing the E1·Mg2+ state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca2+ regulation in heart (for reviews, see, for example, refs8–10), and seems to play an important role in muscle-based thermogenesis11. We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.