To gain insight into the interactions between fengycin and skin membrane lipids, mixed fengycin/ceramide monolayers were investigated using atomic force microscopy (AFM) (monolayers supported on mica) and surface pressure-area isotherms (monolayers at the air-water interface). AFM topographic images revealed phase separation in mixed monolayers prepared at 20 degrees C/pH 2 and composed of 0.25 and 0.5 fengycin molar ratios, in the form of two-dimensional (2-D) hexagonal crystalline domains of ceramide surrounded by a fengycin-enriched fluid phase. Surface pressure-area isotherms as well as friction and adhesion AFM images confirmed that the two phases had different molecular orientations: while ceramide formed a highly ordered phase with crystalline chain packing, fengycin exhibited a disordered fluid phase with the peptide ring lying horizontally on the substrate. Increasing the temperature and pH to values corresponding to the skin parameters, i.e., 37 degrees C/pH 5, was found to dramatically affect the film organization. At low fengycin molar ratio (0.25), the hexagonal ceramide domains transformed into round domains, while at higher ratio (0.5) these were shown to melt into a continuous fengycin/ceramide fluid phase. These observations were directly supported by the thermodynamic analysis (deviation from the additivity rule, excess of free energy) of the monolayer properties at the air-water interface. Accordingly, this study demonstrates that both the environmental conditions (temperature, pH) and fengycin concentration influence the molecular organization of mixed fengycin/ceramide monolayers. We believe that the ability to modulate the formation of 2-D domains in the skin membrane may be an important biological function of fengycin, which should be increasingly investigated in future pharmacological research.