To compare the relative efficacy of trabeculectomy surgery with 90-s and 5-min intraoperative exposure time to 5-fluorouracil. This was a retrospective, non-randomized comparative study. 41 eyes of 33 consecutive patients in the study group were compared to 40 eyes of 30 consecutive patients in the historical control group. Both groups were exposed to 5-fluorouracil (50 mg/ml) during trabeculectomy surgery. The exposure time was 90 s for the study group and 5 min for the control group. Three criteria were used to define surgical success: IOP (intraocular pressure) less than 21 mmHg; IOP less than 21 mmHg with more than 30% reduction in pressure; and IOP less than 15 mmHg with more than 30% reduction. The number of antiglaucoma medications, visual acuity, complications, and interventions were recorded at regular intervals. Mean preoperative IOP was 21.6 +/- 4.8 in the 90-s group and 21.2 +/- 4.9 in the 5-min group. Mean follow-up was 28.2 +/- 5.1 months in the 90-s group and 48.0 +/- 4.9 months in the 5-min group. During the first 30 postoperative months, there were no statistically significant differences in IOP and requirement for antiglaucoma medications between the two groups. The 90-s group had shorter survival rates using the 1st success criteria, but no significant difference was detected when the more stringent 2nd and 3rd criteria were applied. Choroidal effusions were significantly less common in the 90-s group (P = 0.0076). The results of this small study suggest that a 90-s application of 5-fluorouracil may be as effective as a 5-min one in trabeculectomy.