We report the effect of macromolecular crowding on encapsulation efficiency of fluorescently labeled poly(ethylene glycol) (PEG) and dextran polymers within individual giant lipid vesicles (GVs). Low concentrations of the fluorescently labeled polymers (82 nM to 186 pM) were mixed with varying concentrations of nonfluorescent polymers that served as crowding agents during vesicle formation by gentle hydration. Encapsulation efficiency of the fluorescently labeled polymers in individual GVs (EEind) was determined via confocal fluorescence microscopy. EEind for high molecular weight polymers (e.g., fluorescein isothiocyanate (FITC)-dextran 500 and 2000 kDa) increased substantially in the presence of several weight percent unlabeled PEG or dextran. For example, when 0.24 microM FITC dextran 500 kDa was encapsulated, addition of 3% PEG 8 kDa improved the mean concentration in the GVs from 0.14 microM (+/-50%) to 0.24 microM (+/-12%). Light scattering data indicate reduced hydrodynamic radii for polymers as a function of increasing polymer concentration, suggesting that the improvements in EEind result from polymer condensation due to macromolecular crowding. Polymeric cosolutes did not significantly impact EEind for lower molecular weight polymers (e.g., Alexa Fluor 488-PEG 20 kDa), which already encapsulated efficiently (EEind to approximately 1). However, for both the higher and lower molecular weight labeled polymers, cosolutes led to improved uniformity in EEind for vesicles within a batch. Methods for improving the value and homogeneity of EEind for polymeric solutes in lipid vesicles are important in a variety of applications, including the use of vesicles as microreactors and as vehicles for drug delivery.