Aliquots of normal human plasma that had been incubated with tritiated fluorescein were examined for radioactive binding to proteins. Samples were fractionated by polyacrylamide gel electrophoresis (PAGE) and gel filtration. Aliquots removed between zero time and two hours showed no specific radioactive binding peaks by either method. Tritiated fluorescein ran well ahead of all proteins at all times on PAGE and well after the protein eluted from a fractionating column (Sephadex G-75). Ten minutes after intravenous injections, PAGE of plasma from three patients undergoing fluorescein angiography with nonradioactive dye showed the only fluorescent band migrating ahead of all protein bands. These methods failed to demonstrate specific binding of tritiated fluorescein to normal human plasma proteins. We conclude that during angiography, fluorescein exists in plasma as an unbound molecule or is so weakly associated with plasma proteins as to be undetectable by the methods used.