A de novo designed 33-residue polypeptide folds as a compact β-sheet sandwich tetramer in aqueous solution, NMR structural analysis shows that although monomer subunits have the same three-stranded antiparallel β-sheet fold, two equally populated conformational states are identified. Conformational heterogeneity arises from formation of two distinct dimer folds. Each dimer is formed by continuing the monomer β-sheet into a six- stranded sheet similar to that found in α-chemokines. Dimer heterogeneity arises primarily from a two-residue shift in the alignment of interfacial strands. NOE-based conformational modeling has yielded well-defined structures for both dimer types. While the tetramer β-sheet sandwich most probably results from association of hydrophobic surfaces from two amphipathic dimers, dimers could combine to form either two types of homotetramers and/or one heterotetramer composed of both dimer types. Even though interdimer NOEs could not be unambiguously identified to resolve this point, thermodynamic arguments based on observation of equal populations of both dimer types favor formation of heterotetramers.