Purpose: To investigate the effect of latanoprost (LP) on the inward and outward permeability (P<sub>in</sub> and P<sub>out</sub>) of the blood-retinal barrier (BRB). Methods: Four New Zealand white rabbits received topical LP (0.005%) once daily for 3 weeks in one eye and phosphate-buffered saline (PBS) in the fellow eye (topical group). Five New Zealand white rabbits were injected intravitreously with LP (0.1 ml, 0.005%) in one eye and PBS (0.1 ml) in the fellow eye (injection group). In the injection group, vitreous fluorophotometry (VFP) to estimate the P<sub>in</sub> and differential vitreous fluorophotometry (DVF) to estimate the P<sub>out</sub> were performed 60 min after LP was injected. After the baseline measurements, VFP and DVF were performed 60 and 180 min after intravenous injection of sodium fluorescein, respectively. Fluorescein (F) and fluorescein monoglucuronide (FG) concentrations were obtained by DVF, and the F/FG ratio was calculated as an index of the P<sub>out</sub>. Results: In the topical group, there were no significant differences in the P<sub>in</sub> or F/FG ratio between the LP- and the PBS-treated eyes. In the injection group, the P<sub>in</sub> in the LP-treated eyes was significantly higher than in PBS-treated eyes (p < 0.05). There was no significant difference in the F/FG ratio between the two groups. Conclusion: Although we cannot exclude the effect of differences in species, the physiologic effect of LP, which increased the P<sub>in</sub>, was seen in experimental studies. Because antiglaucoma drugs are generally used over an extended period, further clinical studies of the effect of LP on the BRB should be performed in patients who have BRB breakdown, such as in uveitis, and in patients who are pseudophakic and aphakic.