We compared the success rate of filtering surgery of a single 5-minute intraoperative application of mitomycin-C (MMC) or 5-fluorouracil (5-FU). Animal experiment and clinical study were done. In animal study, thirty pigmented rabbits (60 eyes) weighing 2.0 to 2.5 Kg were enrolled. We divided into 4 groups, such as BSS, 5-FU 50 mg/ml, MMC 0.2 mg/ml and MMC 0.4 mg/ml subconjunctival soaked group. Each group consisted of 15 eyes. In each group, 10 eyes was for examination of bleb survival and complications, 3 eyes for light microscopic examination and 2 eyes for electron microscopic examination. Bleb was survived 6.1 days (3 to 13 days) for BSS treated group, 16.3 days (9 to 23 days) for 5-FU 50mg/ml treated group, 32.7 days (17 to 55 days) in MMC 0.2 mg/ml treated group, and 64.4 days (49 to 84 days) in MMC 0.4 mg/ml treated group. Duration of bleb survival was significantly prolonged in 5-FU, MMC 0.2 mg/ml and MMC 0.4 mg/ml group respectively. In clinical study, fifty-five eyes of 40 patients were enrolled. 29 eyes of 21 patients were treated with MMC 0.2 mg/ml and 26 eyes of 19 patients were treated with 5-FU 50 mg/ml. The success rate was 89.7% in MMC 0.2 mg/ml treated group and 84.6% in 5-FU 50 mg/ml treated group at postoperative 3 months, and postoperative 6 months 89.7% in MMC 0.2 mg/ml treated group and 76.9% in 5-FU 50 mg/ml treated group. There was no statistical significance at postoperative 3 months (P > 0.05), but statistical significance at postoperative 6 months (P < 0.05). There was no serious complications using a MMC 0.2 mg/ml or 5-FU 50mg/ml.