OBJECTIVE To compare the aqueous humor concentration of levofloxacin 0.5%, gatifloxacin 0.3% and levofloxacin 0.3% ophthalmic solution after topical dosing in human eyes. METHODS A randomized, double-blind and parallel study was conducted. Senile cataract patients (150 eyes in 150 cases) receiving phacoemulsification in eye center affiliated second hospital of Zhejiang university were randomized into three groups: levofloxacin 0.5%, 50 eyes;gatifloxacin 0.3%, 50 eyes and levofloxacin 0.3%, 50 eyes. Each group was randomized into five sub-groups, with 10 eyes in each sub-group. Each group received either levofloxacin 0.5% or topical gatifloxacin 0.3% or levofloxacin 0.3% preoperatively. One drop of antibiotic was administered every 15 minutes for 4 doses. More than 100 microl aqueous samples were obtained from different sub-groups during surgery, 15.0, 30.0, 60.0, 120.0 and 180.0 min after the last drop, respectively. Then 100 microl aqueous samples were put into test tube by sample injector accurately. Concentration of the antimicrobial agents was determined by the HPLC (high pressure liquid chromatography). Variance analysis was performed to detect differences among the antibiotic treatment groups. The differences between two groups were assumed by t-test. RESULTS Humor concentration of levofloxacin 0.5% (1.61 +/- 0.48), (2.41 +/- 0.80), (2.93 +/- 0.50), (2.56 +/- 0.63), (1.87 +/- 0.88) mg/L was significantly higher than that of gatifloxacin 0.3% (0.70 +/- 0.18), (1.29 +/- 0.54), (1.59 +/- 0.67), (1.41 +/- 0.50), (1.13 +/- 0.28) mg/L and levofloxacin 0.3% (0.55 +/- 0.39), (1.15 +/- 0.42), (1.38 +/- 0.49), (1.02 +/- 0.33), (0.55 +/- 0.31) mg/L at the same period after the last drop (F = 23.64, 12.82, 21.13, 25.00, 12.22;P < 0.05). CONCLUSIONS Topically applied levofloxacin 0.5% had the higher aqueous humor drug level than gatifloxacin 0.3% and levofloxacin 0.3%.