The kinetics of the intraocular penetration of topically applied fluorescein was analyzed by a new method. To the time-courses of fluorescein concentration changes in the aqueous, the bi-exponential equation of Jones-Maurice for a two-compartment model was fitted by a method of least squares. The initial amount of the dye in the cornea divided by the anterior chamber volume, Mo/Va, the cornea-aqueous transfer coefficient in reference to the corneal volume, Kc.ca, and the loss rate of the dye from the anterior chamber, ko, were treated as unknown independent parameters, and they were estimated directly from the concentration changes in the anterior chamber. In the instillation experiments, 20 microliter of phosphate buffered 30% fluorescein solution was instilled in both eyes of 11 normal young subjects and the eyes were washed thoroughly after 5 minutes. The following values were obtained: kc.ca: 4.8 +/- 0.5 X 10(-3) min-1 (Mean +/- SD), ko: 0.78 +/- 0.09 X 10(-2) min-1, and Mo/Va: 0.20 +/- 0.25 micrograms ml-1. The right-left ratio averaged 1.01 +/- 0.05 in kc.ca and 1.02 +/- 0.05 in ko. A similar analysis was carried out in 12 normal young subjects, where the dye was administered to the cornea by ionotophoresis and the results were as follows: kc.ca: 4.7 +/- 0.6 X 10(-3) min-1, ko: 0.83 +/- 0.19 X 10(-2) min-1, and Mo/Va: 0.15 +/- 0.16 micrograms ml-1. The right-left ratio averaged 0.93 +/- 0.12 in kc.ca and 1.02 +/- 0.18 in ko. The anterior chamber volume, Va, was determined by the method of Johnson et al. and it averaged 202 microliter. The aqueous flow rate, i.e., ko.Va, was 1.61 microliter min-1.