A novel HPLC methodology for the study of gramicidin A reconstituted in model membranes has been tested in comparison with circular dichroism data. It is shown that this chromatographic technique not only corroborates most of the recent spectroscopic results but allows one to explain them in terms of mass fractions of different actual conformational species of GA in the phospholipid assemblies. In particular, the dependence of the inserted peptide configuration on the organic solvent and other parameters involved in the 'history' of the sample preparation and handling has been analyzed by HPLC in two phospholipid model systems: small unilamellar vesicles and micelles. Moreover, a slow conformational transition of GA towards a beta 6.3-helical configuration, accelerated by heat incubation, has been also chromatographically visualized and quantitatively interpreted.