OBJECTIVE Permeability (Pdc) to sodium fluorescein (F) is a characteristic of the barrier function of the corneal epithelium. The repeatability of several in vivo fluorophotometric methods used to measure permeability in humans remains largely undocumented. This study examines the repeatability of a method based on topical instillation of a single drop of F for the quantitative assessment of Pdc. METHODS Nine healthy subjects with no history of ocular disease provided 1 (n = 1), 2 (n = 1), or 3 (n = 7) repeated measurements of each eye at successive visits. After making 3 baseline fluorescence scans centrally through the tear film and cornea, 2 microliters of 0.35% F were instilled and 10 fluorescence scans were obtained at approximately 2-minute intervals immediately after instillation. Subsequently, the eyes were rinsed three times with nonpreserved saline and four additional scans were performed. RESULTS Pdc was calculated by dividing the baseline-corrected postrinse stromal fluorescence by the time integral of the tear film fluorescence calculated over the 20-minute exposure period. After applying a logarithmic transformation to the Pdc estimates, a mixed-model analysis was used to assess measurement repeatability. On the Pdc scale, there is an estimated 95% chance that a second measurement could be as much as 2.88 times higher or 0.35 times lower than a first measurement. CONCLUSIONS This substantial variability between repeated measurements indicates that the single-drop procedure is unreliable for monitoring individual patient changes. However, with careful sample size planning, this technique can be used in population-based research to compare differences in treatment effects between groups of subjects.