Surface-pressure (Pi) and surface-area isotherms as a function of surface area were measured for monolayers of amphotericin B (AmB) and cholesterol mixtures at the air/water interface at 10, 20, and 30 degreesC. 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 expanding effect in monolayers was accompanied by a large Pi-A hysteresis and a positive excess of free energy of mixing at high Pi. At low Pi, a condensing effect was observed with the most significant deviation from ideality occurring at a mole fraction of AmB (X-AmB) of 0.67. Free energy calculations revealed a condensing effect at low Pi and an expanding effect at high Pi except at 30 degreesC, where a condensing effect was observed for X-AmB around 0.33. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degreesC exhibited Pi-A isotherms which obey van der Waals equation of state, with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from mixtures of AmB and cholesterol with a 2:1 stoichiometry. (C) 2004 Elsevier Inc. All rights reserved.