The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded beta-sheet segment which connects two globular domains. Although this single-layer beta-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability. Here we report the engineering of OspA variants that contain larger single-layer beta-sheets (comprising five and seven beta-strands) by duplicating a beta-hairpin unit within the beta-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer beta-sheets are formed as designed, and amide hydrogen-deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the beta-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer beta-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.