Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50-60 degrees; (ii) continuous alpha-helix tilted 28 degrees relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes approximately 20 degrees angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solid-state NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.