Mixed monolayers of distearoylphosphatidylcholine (DSPC) and a poly(ethylene glycol)-(PEG)-grafted distearoylphosphatidylethanolamine with a PEG molecular weight of 2000, DSPE-PEG2000, spread on phosphate-buffered saline (PBS) were used as models of bio-non-fouling membrane-mimetic surfaces in order to visualize the lateral distribution of PEG2000-phospholipid in the host phospholipid matrix. Epifluorescence microscopy (EFM) was used to locate DSPE-PEG2000 molecules in the DSPC matrix by detecting the fluorescence from a fluorescein fluorophore attached to the distal end of the PEG2000 chain. Comparative analysis of surface pressure-area isotherms and EFM images revealed that DSPE-PEG2000 mixes nonideally with DSPC in monolayers on a PBS subphase. A transition from a phase-separated monolayer to a homogeneous mixture was observed with increasing surface pressure and PEG content. The effect of nonideal mixing behavior of DSPE-PEG2000 on its lateral distribution in the DSPC matrix was interpreted in terms of excluded volume interactions between the PEG2000 chains and a mismatch in the tilt of aliphatic chains on DSPC and DSPE-PEG2000 molecules.