The conformational properties of protein fragments have been widely studied as models of the earliest initiation events in protein folding. While native-like α-helices and β-turns have been identified, less is known about the factors that underly β-sheet formation, in particular β-hairpins, where considerably greater long-range order is required. The N-terminal 20 residue sequence of native ferredoxin I (from the blue-green alga Aphanothece sacrum ) forms a β-hairpin in the native structure and has been studied in isolation by NMR and CD spectroscopy. Local native-like interactions alone are unable to stabilize significantly a folded conformation of the 20-residue fragment in purely aqueous solution. However, we show that the addition of low levels of organic co-solvents promotes formation of native-like β-hairpin structure. The results suggest an intrinsic propensity of the peptide to form a native-like β-hairpin structure, and that the organic co-solvent acts in lieu of the stabilizing influence of tertiary interactions (probably hydrophobic contacts) which occur in the folding of the complete ferredoxin sequence. The structure of the isolated hairpin, including the native-like register of interstrand hydrogen bonding interactions, appears to be determined entirely by the amino acid sequence. The solvent conditions employed have enabled this intrinsic property to be established.