A uniquely structured 65 amino acid helix-loop-helix＇-loop-helix " three-alpha-helix bundle, alpha(3)-1, was designed and chemically synthesized, using the crystallographically characterized three stranded coiled coil "Coil-Ser", as a starting point. The circular dichroism spectrum of alpha(3)-1 has a typical alpha-helical signature, with a [theta](222) = -22 600 deg.cm(2).dmol(-1), indicating a 80.5% alpha-helical content. Sedimentation equilibrium analytical ultracentrifugation revealed that alpha(3)-1 is monomeric in solution. Consistent with the design parameters, the fluorescence emission maximum of the unique hydrophobic core tryptophan residue occurs at 324 nm. The evaluated Delta G(H2O) based on reversible guanidine hydrochloride denaturation is -4.6 +/- 0.3 kcal.mol(-1) (m = 2.2 +/- 0.2 kcal.mol-(1).M-1) as measured by CD spectroscopy. The amide-aromatic region of the H-1-NMR spectrum of alpha(3)-1 illustrates excellent chemical shift dispersion and resolution. All 35 expected methyl correlations are accounted for in the C-13-HSQC spectrum, providing stringent evidence for the existence of a native-like hydrophobic core. The monomeric nature of alpha(3)-1 should facilitate NMR structural studies and kinetic protein folding analysis of the current design, and on future variants with engineered binding sites. The utility of this single-chain three-a-helix bundle framework for expanding the range of biochemical cofactors bound in maquettes is being explored.