The 26-residue toxin from Staphylococcus aureus, δ-Hemolysin, is thought to act by traversing the plasma membrane. The structure of this peptide, in methanol solution, has been investigated by using high-resolution NMR in combination with molecular dynamics calculations. The1H NMR spectrum has been completely assigned, and it is shown that residues 2–20 form a relatively stable helix while the residues at the C-terminal end appear to be more flexible. The structures were calculated only from nuclear Overhauser effect data and standard bond lengths. It is shown that the results are consistent with3JNH-α CHcoupling constants and amide hydrogen exchange rates.