A prospective, randomized, double-masked and placebo-controlled study was performed to compare the effects of a single 5-minute intraoperative exposure to aclacinomycin (AMC) 0.4 mg/ml or 0.8 mg/ml with control eyes treated with saline solution on the success of glaucoma filtration surgery in 26 rabbits. Intraocular pressure (IOP), bleb survival, fistula patency and complications were evaluated. The results showed that IOP in the eyes treated with AMC was significantly lower than that in the control eyes from days 5-40 in the 0.4 mg/ml group and from days 5-20 in the 0.8 mg/ml group. The bleb survival lasted significantly longer in the two treated groups than in the control group and in the AMC 0.4 mg/ml group than in the AMC 0.8 mg/ml group. At 40 days, the rate of sclera fistula occlusion was 0% in the AMC 0.4 mg/ml eyes, 43.8% in the AMC 0.8 mg/ml eyes, and 100% in the control eyes. Significant complications, such as anterior chamber inflammation, hyphema, moderate and severe corneal haze, dense corneal neovascularization and mild cataract occurred only in the eyes treated with AMC 0.8 mg/ml. The results indicated that intraocular treatment with AMC at a dose of 0.4 mg/ml had a markedly beneficial effect on IOP, bleb appearance and fistula patency after experimental filtration surgery in rabbits.