The morphology of amphotericin B monolayers spread at the air/water interface was investigated at the microscopic level with a Brewster angle microscope. The images were recorded simultaneously with the surface pressure (pi)/area (A) isotherms. The transformation from the domain-like structure to homogeneous solid phase was observed upon monolayer compression. Quantitative analysis was made from the measurements of relative reflectivity during monolayer compression/decompression. A 3-fold increase in relative monolayer thickness throughout the compression from the expanded to a condensed state is observed and related to orientational changes of the monolayer molecules from a horizontal to a vertical position.