Calcium ions (Ca2+) have an important role as secondary messengers in numerous signal transduction processes(1-4), and cells invest much energy in controlling and maintaining a steep gradient between intracellular (similar to 0.1-micromolar) and extracellular (similar to 2-millimolar) Ca2+ concentrations(1). Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca2+-ATPases (PMCAs), are key regulators of intracellular Ca2+ in eukaryotes(5-8). They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca2+ levels in the nanomolar range as well as micromolar-range Ca2+ transients generated by cell stimulation(7). Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2: 1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural-model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca2+ concentration, where steep activation occurs.