A simple, selective method is described for separation of more than 10 gramicidin components on Spherisorb ODS B, 5 mum, 250 x 4.6 mm I. D. column, maintained at 50 degreesC. The mobile phase comprised methanol-water (71:29) at a flow rate of 1.0 mL min(-1). Detection was by UV at 282 nm. Valine gramicidins A, B and C were very well separated from the isoleucine gramicidins A, B and C. Four new gramicidin components were also resolved and their structures determined by liquid chromatography/mass spectrometry. The names 10-methionine valine gramicidin C, 4-methionine valine gramicidin A, valine gramicidin A hydroxypropyl and isoleucine gramicidin A hydroxypropyl were proposed. Robustness of the liquid chromatography method was evaluated by performing a full factorial design experiment. The method also showed good repeatability, linearity and sensitivity.