A prospective, randomized study was performed to examine the effects of subconjunctival retention of 5-fluorouracil (5-FU) microparticles or 5-FU microparticles combined with mitomycin-C (MMC) on the success of trabeculectomy procedure in 32 New Zealand white rabbits. Drug-loaded microparticles were prepared using a biodegradable polymer, 50:50 ploy (D,L-lactide-co-glycolide) (50:50, PLG, MW 9000), by an oil-in-oil emulsification/solvent extraction technique. Each rabbit underwent trabeculectomy on both eyes, then one of the 5-FU and/or MMC preparations was placed at the site of the filtering surgery intraoperatively in the right eye, while the left eye was used as a control. The rabbits were randomly allocated to one of four treatment groups: group 1 rabbits received 5% 5-FU microparticles 10 mg; group 2 rabbits received 10% 5-FU microparticles 10 mg; group 3 rabbits received 10% 5-FU microparticles 5 mg and MMC 0.01 mg (5-FU/MMC); and group 4 rabbits received MMC 0.02 mg. Postoperatively, intraocularpressure (IOP), bleb survival, complications and IOP dynamics were compared during a follow-up period of 42 days. The results showed that IOPs were significantly lower in the eyes that received groups 3 and 4 treatments, while the eyes that received low concentrations of 5-FU microparticles resulted in no difference in IOPs as compared with those of controls. At 42 days, blebs were present in 100% of the eyes treated with 5-FU/MMC and MMC, but 0% of the eyes treated with 5-FU microparticles. The blebs in the eyes treated with MMC 0.02 mg were thinner, and significant complications (endophthalmitis, transient corneal opacification and neovascularization) occurred. In addition, the IOP dynamics study revealed that the eyes treated with 5-FU/MMC and MMC resulted in less IOP spikings after intravenous infusion of 0.9% NaCl solution. Our study suggested that the use of 5-FU microparticles in promoting the success of trabeculectomy in rabbits is dose-dependent, and its effect is less potent than a single intraoperative application of MMC.