The solution structure of a de novo designed disulfide-bridged two-alpha-helix peptide that self-assembles to form a 2-fold symmetric four-alpha-helix bundle protein (alpha'-SS-alpha')(2) has been solved by NMR spectroscopy. The 33-residue peptide, (alpha'-SH), that is the basic building block of the bundle has been recombinantly expressed. The three-dimensional structure of the asymmetric unit of the bundle has been determined using interproton distance restraints derived from the nuclear Overhauser effect (NOE), covalent torsion angle restraints derived from three bond scalar coupling constants, and longer range angular restraints derived from residual dipolar couplings. The covalent alpha'-SS-alpha' unit forms a pair of parallel alpha-helices that use heptad a-, d-, e-, and g-side chains to form a hydrophobic core extending the length of the molecule. The distribution of polar and nonpolar side chains on the surface of alpha'-SS-alpha' structure is asymmetric. The hydrophilic face is comprised of glutamate and lysine side chains, while the opposite face is comprised of leucine, isoleucine, phenylalanine, tryptophan, and neutral histidine side chains. Equilibrium sedimentation analysis, size-exclusion chromatography, pulsed field gradient translation diffusion measurements, and a rotational correlation time derived from N-15 NMR relaxation studies all indicate that the covalent alpha'-SS-alpha' unit forms a noncovalent dimer, (alpha'-SS-alpha')(2), in solution. The structure confirms many expected design features and illuminates an apparent dichotomy of structure where the helical interface of the disulfide bridged two-alpha-helix peptide appears nativelike while the adjacent, noncovalent interface shows non-nativelike behavior. Available evidence indicates the four alpha-helix bundle can adopt either an anti or syn topology. The structure is discussed with respect to the potential origins of conformational specificity and nativelike protein structure.