We report encapsulation of polymers and small molecules within individual giant lipid vesicles (GVs; 3-80 mu m), as determined by confocal fluorescence microscopy. Polymer-bound or free dyes were encapsulated within GVs by including these molecules in the aqueous solution during vesicle formation via gentle hydration. Encapsulation efficiencies of individual GVs (EEind) were determined from the fluorescence intensity ratio inside vs outside the vesicle. EEind varied considerably from vesicle to vesicle, with interior solute concentrations for GVs within the same batch ranging from much less than to slightly more than the initial concentration. The majority of GVs had high internal concentrations of polymer or small-molecule encapsulants equal to or slightly greater than the external concentration. EEind decreased for high molecular weight polymers (e.g., dextran 500 000), but was relatively insensitive to the GV diameter, membrane composition, or incubation temperature in our experiments. Knowledge of EEind is important for quantitative evaluation of reactions occurring within GVs (e.g., enzymatic processes) and for optimizing encapsulation conditions.