Following injections of Evans blue (1-200 mg/kg) into rabbits, polyacrylamide gel disc electrophoresis showed that Evans blue binds to two protein fractions. The greater part was bound to albumin and the remainder to a plasma protein in the postalbumin fraction. Unbound Evans blue was present in each plasma sample analyzed. Attempts to liberate the dye from the coloured areas of the aorta and common carotid arteries by tissue electrophoresis failed unless very high concentrations of Evans blue were used. This indicates that at the concentrations used by many investigators areas dyed by Evans blue may not be equated with the presence of diffusible protein-dye complexes.