Amphotericin B has been separated into five components by the use of thin-layer chromatography (TLC) with only methanol as the developing solvent. Each TLC component was then subjected to high-performance liquid chromatography. Besides the two major components found in other studies, amphotericin B was also found to contain five other significant heptaenes, one tetraene, two fluorescent polyenes (pentaene and heptaene), and at least five other significant components which had appreciable absorbance at approximately 305 nm. In addition, amphotericin B was found to exhibit as in a previous study by other workers, only a trace amount of fluorescence.