It has been demonstrated by two-dimensional NMR cross-relaxation spectroscopy that gramicidin A exists in dimethylsulfoxide/acetone solution in random coil form. This contradicts earlier conclusions by Hawkes et al. [Hawkes, G. E., Lian, L. Y., Randall, E. W., Sales, K. D. & Curzon, E. H. (1987) Eur. J. Biochem. 166, 437-445] that were based on the interpretation of vicinal proton coupling constants.