A method has been developed for the separation of the filipin complex components by gradient-elution high performance liquid chromatography (HPLC). The elution order for the major filipin components (filipins I-IV) was established by first isolating the component fractions by thin-layer chromatography. Each component fraction was then subjected to gradient HPLC. The order of elution for the major filipin components was from first to last : III, IV, II and I. The unexpected reversal in the elution order for filipin III and IV may be evidence that the two filipins are stereoisomeric at the C-1' position. Finally, gradient elution HPLC was used to compare various preparations of filipin. In addition, the technique has been applied to other preparations of polyene antibiotics which have structures similar to that of filipin.