Objective It is to discuss the effects of topical prazosin and pilocarpine on uveoscleral(Fu) outflow in rabbits. Methods Sixteen rabbits were at random divided into the control group(5 rabbits, only topical application of normal saline in right eye of each rabbit), prazosin(PZ)-treated group(6 rabbits, only prazosin eyedrop 0.1% in right eye of each one) and pilocarpine(PC)-treated group(5 rabbits, pilocarpine eyedrop 1% in each right eye). Intraocular pressure (IOP) of bilateral eyes of each rabbit were measured before and 1hr later after eye drop topical application. And the bilateral eyes were perfused with Fluorescein-isothiocyanate bovine serum albumin (FITC-BSA) as the tracer into anterior chambers of each rabbit for 30 min at 90 min after topical treatment. Then the rabbits were killed to measure Fu. Results IOP of PZ-treated eye decreased 0.71±0.07kPa in 1 hour later after PZ application. IOP of PC-treated eye decreased 0.70±0.08kPa in 1 hour later after PC application. The average value of Fu was 0.176±0.048 μl/min in control eye. The average value of Fu in PZ-treated eye was 0.339±0.018 μl/min. The average value of Fu in PC-treated eye was 0.123±0.022 μl/min. Conclusion Both of PZ and PC can decrease IOP in rabbits following topical application. Topical PZ can increase Fu in rabbits and this is one of the mechanisms of PZ-induced IOP decrease. Topical PC can prevent Fu. PC decrease IOP not through uveoscleral pathway. This study demonstrates that uveoscleral pathway plays an important role in aqueous humor drainage. PZ may be a novel drug for decreasing IOP. FITC-BSA is an effective tracer for studying uveoscleral pathway.