OBJECTIVE To study the pharmacokinetics of levofloxacin hydrochloride in-situ gel in aqueous humors of rabbit eyes.METHODS Levofloxacin hydrochloride concentration in aqueous humors of rabbit eyes at different sampling time was determined after the dose of in-situ gel by RP-HPLC. The mean aqueous humors concentration-time curve of levofloxacin hydrochloride was discribed and pharmacokinetic parameters were calculated.RESULTS The absorption and elimination of levofloxacin hydrochloride were fitted to a linear model with the main pharmacokinetic parameters as follows: t_1/2(ke), MRT, t_max,ρ_max, AUC_0-τ and AUC_0-∞ were (118.76±7.21) min, (208.40±13.46) min, (34.07±5.02) min, (2.15±0.075) mg·L^-1, (432.05±13.55) mg·min·L^-1 and (501.52±25.24) mg·min·L^-1,respectively. CONCLUSION The release of levofloxacin hydrochloride in the eyes can be significantly prolonged by the in-situ gel.