PURPOSE: This study was performed to separately assess the aqueous flow applied with hyaluronic acid, and the behaviour of hyaluronic acid itself on the ocular surface. METHODS: Two different fluorescent dyes, fluorescein sodium dissolved in 0.1% hyaluronic acid (HA) solution and 0.1% fluorescein conjugated with hyaluronic acid (F-HA) dissolved in saline, were used. A volume of 20 mul of tested solution was applied to the eye of 10 healthy volunteers. Fluorescein sodium dissolved in saline served as a control. The fluorescent intensity of the precorneal tear film was measured at the central cornea every minute for 10 min. The turnover rate was calculated using the equation that plots fluorescent intensity against time in a semilog plot and expressed as %/min. RESULTS: Turnover rates of topically applied 0.1% F-HA, 0.1% HA and saline were 8.1 (SD 3.6)%/min, 21.6 (2.8)%/min, and 31.0 (3.7)%/min, respectively. The turnover rate of F-HA was significantly lower than those of HA and saline (p = 0.00012 and p = 0.00000022, respectively; Mann-Whitney test). The turnover rate of HA was significantly lower than that of saline (p = 0.00001; Mann-Whitney test). CONCLUSION: Our results indicate that the bulk aqueous flow applied with HA and the turnover of HA itself are different. HA molecules may adhere to the ocular surface by surface-chemical and/or biochemical properties. The long retention time of HA on the ocular surface may explain the mechanism in which hyaluronic acid has been shown to enhance tear film stability for a few hours.