Recently, our group was the first to apply the technique of single molecule fluorescence imaging toward homogeneous phospholipid monolayers at the air-water interface to study the lateral diffusion properties within-these systems at the single molecule level.1 Here, we present measurements on Langmuir monolayers of amphiphiles at the air-water interface of a specific heterogeneity to explore problems of hindered lateral diffusion of biomolecules observed in complex biomembranes. The lateral mobility of fluorescence-labeled phospholipids in mixtures of phospholipids and lipopolymers was investigated via single molecule tracking at different lipopolymer molar concentrations. In agreement with recent fluorescence recovery after photobleaching experiments,2 we found that the diffusion behavior of phospholipids within polymertethered monolayers is characterized by different regions depending on the lateral mobility of lipopolymer molecules. At a low lipopolymer molar concentration, the diffusion coefficient of phospholipids is independent of the concentration of tethered lipids (lipopolymers), thereby showing no signs of obstructed diffusion. Clear signs of obstructed diffusion are observed, however, if polymer chains of adjacent lipopolymers interact with each other. Our experiments showed that single molecule fluorescence imaging is a powerful experimental tool to study obstructed diffusion in model membranes.