The suggested procedures of the British Pharmacopoeia for the microbiological assay of the polyene antifungal antibiotics amphotericin B, candicidin and nystatin have been compared. A simpler procedure has been examined and shown to be suitable for the microbiological assay of all three antibiotics. The increased sensitivity of the assay is due to the enhanced solubility of the antibiotics in the diluents used for preparing the assay solutions. The assay has been shown to be accurate and precise.