We describe an integrated fluorescence, polarized fluorescence, and Brewster angle microscope/ Langmuir trough assembly. This apparatus was specifically designed for the study of lung surfactant (LS) monolayers, and is well suited for the study of other lipid/protein monolayer systems. The apparatus can be operated simultaneously in both the fluorescence and Brewster angle modes under a wide range of conditions, including physiological subphases and temperatures. The combination of information obtained from these microscopy techniques facilitates the identification of the composition of coexisting phases, allows us to systematically study the effects of specific proteins on lipid monolayers, and eliminates possible artifacts inherent to fluorescence. To demonstrate this system we present images of mixed monolayers of the anionic and unsaturated lipid components of LS and show that synergistic interactions between certain LS proteins and these lipids hold the key to the proper functioning of LS monolayers.