The conformation of pituitary adenylate cyclase activating polypeptide with 27 residues (PACAP27) has been determined by two-dimensional NMR and CD spectroscopies and distance geometry in 25% methanol. Residues 9-20 and 22-25 have well-defined conformations but other residues do not show ordered conformations. The conformation of residues 9-20 is composed of three distinct regions of beta turn-like conformation (residues 9-12), alpha helix (residues 12-14) and the looser helical conformation (residues 15-20), while residues 22-24 form alpha helix. PACAP27 has a 2 helices separated by a disordered region similar to a VIP analog reported by Fry et al. but is distinct from the VIP analog in the position of the first helix, which is shifted by 2 residues toward the C-terminus, and in the form of the second helix [Fry, D.C., Madison, V.S., Bolin, D.R., Greeley, D.N., Toome, V. and Wegrzynski, B.B. (1989) Biochemistry 28, 2399-2409].