The antimycotic activity of amphotericin B depends on its ability to make complexes with cell membrane sterols. Surface pressure (pi) as a function of surface area (A) and pi-A hysteresis were measured for monolayers of amphotericin B/cholesterol mixtures on the water/air interface. Specific area per molecule of amphotericin B and free energy of mixing were calculated as a function of concentration of amphotericin B. When chloroform/methanol was used as a spreading solvent, the pi-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The mean molecular area of the mixed monolayers was significantly higher than the calculated sum of the molecular areas of the pure components. This expanding effect was accompanied by a large pi-A hysteresis and a positive excess of free energy of mixing at high pi. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degreesC exhibited pi-A isotherms with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from molecules of amphotericin B and cholesterol with a 2:1 stoichiometry. At this ratio, amphotericin B and cholesterol form ion channels in lipid bilayers with conductance of 4-400 pS. These results provide a better understanding of the biological activity of amphotericin B. Artificial amphotericin B/cholesterol ion channels having large conductance could be useful in nanotechnology.