The structure of human corticotropin releasing factor (hCRF) has been determined by proton nuclear magnetic resonance (1H NMR) in a mixed-solvent system of 66% trifluoroethanol/34% H2O at pH 3.8 and 37°C. Nearly complete resonance assignment was achieved by using standard two-dimensional methods. Distance restraints for structure calculations were obtained by qualitative analysis of intra- and interresidue nuclear Overhauser effects. Structures were obtained from the distance restraints by distance geometry, followed by refinement using molecular dynamics and were completed with amide hydrogen exchange data. The structure of hCRF in this solvent comprises an extended N-terminal tetrapeptide connected to a well-defined α-helix between residues 6 and 36. The first half of the α-helix (residues 6–20) is clearly amphipathic. The five carboxy-terminal residues are predominantly disordered.