Abstract: AIM　To study the pharmcokinetics of fluconazole (FCZ) in the aqueous humors and tears of New Zealand white rabbits following topically applied in-situ-forming gels of 0.5% FCZ (ISG-FCZ) and 0.5% FCZ eye drops to rabbit eyes. METHODS　The rabbit tears and aqueous humors were obtained and quantified at different times after topically applying single dose of ISG-FCZ and FCZ eye drops to the rabbit eyes. The drug levels were assayed by megabore capillary gas-liquid chromatography with nitrogen-selective detector. The pharmacokinetic parameters were calculated with nonlinear least square method　with　computer.　RESULTS FCZ concentrations in rabbit tears within 180 min after applying ISG-FCZ were found to be significantly higher compared with those of FCZ eye drops. The FCZ levels in the aqueous at 10, 30, 60, 90 and 120 min after applying ISG-FCZ are significantly higher compared with FCZ eye drops. The time for arriving peak concentration (T_max) and the area under concentration (AUC)-time curve as well as the half-life (T_1/2) of FCZ in rabbit aqueous humors were markedly higher after application of ISG-FCZ. The peak levels in the aqueous humors showed no difference between the two groups. CONCLUSION　The ISG-FCZ, which upon exposure to physiological conditions will shift the gel phase, can significantly extend FCZ release and increase the precorneal residence time of the drug, thus enhance ocular bioavailability compared with FCZ eye drops.