pi-A isotherms of mixed monolayers composed of cholesterol and amphotericin B (AmB) spread on aqueous buffers of various pH and temperatures show the existence of interactions between the two components, which are more pronounced when the mole fraction of AmB is 0.7. As a consequence of the interactions, the excess areas and excess free energies of mixing are negative at low surface pressures and positive at high surface pressures. These results suggest that negative deviations of the additivity rule are due to the formation of a hydrogen-bonded AmB-cholesterol complex in which AmB molecules are oriented horizontally at the interface and cholesterol molecules lie vertically. The positive excess areas of mixing at high surface pressures could be due to AmB being less desorbed in the substrate by composition-dependent van der Waals interactions between the apolar moieties of the components, bath oriented in this situation in a vertical position at the A/W interface.