Conformational study of gramicidin S and N,N′-diacetyl gramicidin S has been carried out by a number of techniques. Optical rotatory dispersion measurements showed both cyclodecapeptides to possess similar conformations in different solvents. Quantitative infra red studies in dilute chloroform solutions lead to the inference that there are ca. six intramolecular hydrogen bonds in the N,N′-diacetyl derivative. Of these deuterium exchange rates by proton magnetic resonance measurements indicate four strong H-bonds due to the Val and Leu NH groups and two weaker bonds due to the Orn α-NH groups. The NH-C αH proton coupling constants show the protons of this fragment to be trans in Val, Orn and Leu residues and gauche in D-Phe residues. The data obtained provide unequivocal proof of the Hodchkin-Oughton-Schwyzer model of gramicidin S, for which φ and ψ coordinates are given.