Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such molecules can, for example, mimic the ability of natural prototypes to bind to specific target macromolecules but resist degradation by proteases. We have previously shown that oligomers containing mixtures of alpha- and beta-amino acid residues ("alpha/beta-peptides") can mimic the alpha-helix secondary structure, and that properly designed alpha/beta-peptides can bind to proteins that evolved to bind to alpha-helical partners. Here we report fundamental studies that support the long-range goal of extending the alpha/beta approach to tertiary structures. We have evaluated the impact of single alpha ->beta modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several alpha-helical segments packed around a small hydrophobic core. We examined alpha ->beta substitution at four solvent exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the beta(3) homologue of the natural a residue and a cyclic beta residue were evaluated. All alpha ->beta(3) substitutions caused significant destabilization of the tertiary structure as measured by variable-temperature circular dichroism, although at some of these positions, replacing the beta(3) residue with a cyclic beta residue led to improved stability. Atomic-resolution structures of four VHP analogues were obtained via quasiracemic crystallization. These findings contribute to a fundamental alpha/beta-peptide knowledge-base by confirming that beta(3)-amino acid residues can serve as effective structural mimics of homologous alpha-amino acid residues within a natural tertiary fold, which should support rational design of functional alpha/beta analogues of natural poly-alpha-peptides.