The detailed conformation of a tetrapeptide of tropoelastin, t-Boc-L-Val1-L-Pro2-Gly3-Gly4-OMe in CDCl3, has been obtained from a combined analysis of 1H NMR spectra and conformational energy calculations. The observations of Gly3 and Gly4 methylene protons as ABX spin systems indicate a fixed conformation similar to a cyclic peptide stabilized by hydrogen bond formation. Temperature dependence and solvent perturbation of NH protons and conformational energy calculations each showed the presence of a β-turn, a ten atom hydrogen-bonded ring involving the Gly4NH and Val1C=O, and a segment of an antiparallel β-pleated sheet stabilized by a hydrogen bond between the Val1NH and the Gly4C=O. Conformational angles obtained from the observed 3JαCH-NH coupling constants and from conformational energy calculations were in good agreement. The secondary structure of this tetramer is shown to be the same as previously proposed for the high polymer of the tetramer in water at elevated temperature. © 1976, American Chemical Society. All rights reserved.