To compare the effects of three different concentrations of mitomycin C (MMC) as an adjunct therapy to trabeculectomy, we reviewed 26 patients (36 eyes) who underwent trabeculectomy in which MMC was applied intraoperatively. The time of application was 5 minutes, and the concentration of MMC was 0.1 mg/ml (n = 10), 0.2 mg/ml (n = 17) and 0.4 mg/ml (n = 9), respectively. Complete success was defined by intraocular pressure (IOP) of 21 mmHg or less without medication at postoperative 3 months. There was no statistical difference among the three groups in mean age of patients, preoperative IOPs, and the numbers of premedication. The mean IOP at postoperative 3 months was significantly lower in the 0.4 mg/ml group (10.4 ± 5.5 mmHg) than in the 0.2 mg/ml group (16.1 ± 4.7 mmHg) (p < 0.05). The complete success rate in the 0.4 mg/ml group was 100%, which was significantly higher than the rate of 40% obtained with the 0.1 mg/ml group (p < 0.05), but was not significantly different to the rate of 70.6% in the 0.2 mg/ml group (p > 0.05). There was no statistical difference among the three groups in the success rate regardless of medication, and the size and longevity of the filtering bleb. Postoperative hypotony was noted in two eyes of the 0.4 mg/ml group.