The binding of fluorescein was measured in human plasma using four methods: steady-state dialysis, pressure dialysis, equilibrium dialysis, and fluorescence polarization. All methods indicated that fluorescein is approximately 85% bound throughout a wide range of concentrations. Technical difficulties were encountered with the fluorescence polarization method above fluorescein concentrations of 10(-5) g/mL, but below this concentration, the method gave reproducible and reliable results compared with the three other methods. The number of binding sites available in plasma was estimated to be 4 x 10(-3) M, and the dissociation constant was estimated to be 6 x 10(-4) M. These estimates imply that the bound-free ratio of fluorescein in plasma is almost constant at concentrations below 10(-4) g/mL. Fluorescein binding can affect the fluorescent intensity or the rate at which fluorescein penetrates the blood-ocular barriers.