Abstract Monolayers that spread at the air/water interface are considered very useful models of biological membranes because the molecular area of their constituents, the pH and the ionic content of the subphase can be controlled. Spectroscopic information on such systems was obtained by means of a fluorimeter of our fabrication that was specially designed for their study. The technique uses the property of cylindrical symmetry of the monolayers, whereby structural changes of the lipid matrix can be investigated by using a linearly polarized incident light. When the fluorescent probe is photosensitive, lateral diffusion coefficients are measured by monitoring the fluorescence after photobleaching. In this study, bleaching of anthroyloxy derivatives was used in the investigation of lipid phase transitions, of the interaction between ions and acidic lipids and of the complex that forms between phosphatidylglycerol and polymyxin B.