The solution conformation of the 27 residue polypeptide hormone secretin has been investigated by 1H-NMR spectroscopy under conditions where it adopts a fully ordered structure as judged by circular dichroism spectroscopy, namely in an aqueous solution of 40% (v/v) trifluoroethanol. Using a combination of two-dimensional NMR techniques the 1H-NMR spectrum of secretin is completely assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. It is shown that under these conditions secretin adopts a conformation consisting of an N-terminal irregular strand (residues 1-6) followed by two helices (residues 7-13 and 17-25) connected by a 'half-turn' (residues 14-16); the last two residues (26 and 27) are again irregular. This conformation is shown to be very similar to that of glucagon in perdeuterated dodecylphosphocholine micelles and to that of the active 1-29 fragment of growth hormone releasing factor in 30% (v/v) trifluoroethanol: