The extent of complex formation between the polyene antibiotic, amphotericin B, and cholesterol or ergosterol was investigated and a method for a quantitative measurement of the complex formation was developed. The effect of experimental conditions on the magnitude of the amphotericin B-sterol interaction and on the selectivity of this interaction showed that there was only a narrow range of solvent composition in which the differential selectivity of amphotericin B towards these two sterols could be observed.