Amphotericin B is the most important clinically and best defined chemically of the macrolide heptaene antibiotics. A fermentation product of the soil actinomycetes Streptomyces nodosus, amphotericin B binds selectively to ergosterol in the cell membrane of susceptible fungi, inducing changes in permeability that can produce lethal cell injury. Available since 1960, amphotericin B remains the treatment of choice for most serious systemic fungal infections. This review highlights some important aspects of the physicochemical properties of amphotericin B and their utilisation in its quantitative determination in biological fluids. Its mechanism of action is re-appraised in the light of recent results with regard to its effects on the physical and functional properties of synthetic and natural membranes. Attempts to reduce its toxic effects to host cells and to improve its therapeutic index are evaluated. In addition, the therapeutic values of amphotericin B, apart from its antibiotic activity, are discussed.