In a phase I/II study, the new fluorescence marker 5-aminofluorescein covalently bound to human serum albumin (AFL-HSA) was intravenously administered to 10 patients with brain tumor 1 to 4 days before surgery, and AFL-HSA kinetics were determined to assess the optimum timing of dye administration. AFL-HSA was determined in serum and brain tissue by size exclusion chromatography with fluorescence detection. AFL-HSA disposition was quite similar to albumin kinetics with small volumes of distribution (volume of central compartment [V(c)] = 2.4 L; volume of distribution [Vd(β)] = 6.8 L; volume at steady state [V(ss)] = 5.9 L), a low clearance (mean Cl = 16.85 mL/h), and a long elimination half-life (mean T(1/2;β) = 307 hours). Predicted peak concentrations in the peripheral compartment occurred at 80.9 hours, the time point of optimal fluorescence intensity observed in brain tumors during surgery.