Abstract: The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH ≈ 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 m (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of ≈ 8 m. Temperature dependent CD experiments support a ‘cold denaturation’ of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 m HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NHi/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10–G11–T12. The helical structure of melittin obtained at 3.6 m HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.