Phospholamban is a 52 amino acid residue membrane protein involved with the regulation of calcium levels across sarcoplasmic reticulum membranes in cardiac muscle cells. The N-terminal 30 amino acid residues of the protein are largely hydrophilic and include two sites whose phosphorylation is thought to dissociate an inhibitory complex between phospholamban and Ca2+ ATPase. The C-terminal 22 amino acid residues are largely hydrophobic, anchor the protein in the membrane and are responsible for Ca2+ selective ion conductance. Specific interactions between the transmembrane domains stabilize a pentameric protein complex. We have obtained circular dichroism (CD), transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of the full-length protein and have compared these results to those from a 28 residue peptide that includes the transmembrane domain. Both proteins reconstituted into phospholipid membranes are largely alpha-helical by CD and FTIR. Polarized ATR-FTIR measurements show that both the cytosolic and transmembrane helices are oriented perpendicular to the membrane plane with a tilt of 28 (+/- 6) degrees with respect to the membrane normal. This tilt angle is in close agreement to that calculated from a model for the transmembrane domain of phospholamban suggested by mutagenesis and molecular modeling. Phosphorylation does not significantly change the secondary structure or orientation of the protein. The pentameric complex is modeled as a left-handed coiled-coil of five long helices (40 (+/- 3) residues) that extend across the membrane from the lumenal carboxy terminus to the phosphorylation site in the cytoplasm. The helix bundle forms a perpendicular ion pore that may begin at a distance (17 to 29 A) from the membrane surface. Based on the above, we propose a mechanism by which phospholamban regulates Ca2+ levels across membranes that takes into account both its selective ion conductance and inhibitory association with the Ca2+ pump.