The binding to human albumin of fluorescein monoglucuronide, a fluorescent metabolite of fluorescein, was studied using two methods: pressure dialysis and fluorescence polarization. Both methods indicated that fluorescein monoglucuronide binds to human albumin more loosely than fluorescein. The free fraction in human plasma estimated from the dissociation constant and the number of binding sites was in a range from 31 to 37%. Fluorescence of fluorescein was significantly quenched by the albumin binding, but fluorescence of fluorescein monoglucuronide was not affected by albumin. The relative molar intensity of fluorescence between these fluorophores varied from 3.2 to 37.3, depending on the excitation wavelength.