The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.