The final steps in the assembly of rotavirus occur in the lumen of the endoplasmic reticulum (ER). Targeting of the immature inner capsid particle (ICP) to this compartment is mediated by the cytoplasmic tail of NSP4, a non-structural virus glycoprotein located in the ER membrane. To delineate structural and functional features of NSP4, soluble fragments of the cytoplasmic tail have been expressed and purified. Our analysis combines a functional assay for ICP binding with biochemical and CD spectroscopic studies to examine the secondary and quaternary structure. The ICP-binding domain is located within the C-terminal 20 amino acids of the polypeptide. A second region, distinct from this receptor domain, adopts an alpha-helical coiled coil structure and mediates the oligomerization of the virus binding domains into a homotetramer. The domain organization of the cytoplasmic fragments of NSP4 suggests a novel structure for an icosahedral virus receptor protein in which C-terminal binding sites for immature rotavirus particles are connected to an alpha-helical coiled coil stalk which projects from the ER membrane.