The purpose of this study was to determine whether 5-fluorouracil administered as a single intraoperative dose beneath the conjunctival flap during trabeculectomy in patients with glaucoma would be safe and effective. This prospective case series consists of 43 eyes of 43 consecutive patients who underwent trabeculectomy with intraoperative topical administration of 5-fluorouracil. Surgical outcome and success rate of the procedure were evaluated at a 6-month follow-up period. Success was defined as a postoperative intraocular pressure (IOP) of </=21 mm Hg with a minimum 20% drop in IOP from preoperative levels and one Snellen line or less loss of visual acuity, at the end of 6 months. The overall success rate was 85.4%. Mean IOP was 31.4 +/- 11.4 preoperatively and 15.1 +/- 4.6 postoperatively. The mean number of antiglaucoma medications was reduced from 2.8 +/- 0.9 to 0.4 +/- 0.7. Success rates were 69.2% in eyes with high risk for failure and 92.9% in relatively low-risk eyes. A thin and ischemic bleb was characteristic in the immediate postoperative period and remained so in all successful eyes with the exception of five eyes (11.6%) that developed encapsulated blebs. There were no corneal epithelial complications. Intraoperative 5-fluorouracil application directly to the trabeculectomy site appears to be a safe and effective method. It can be a valuable alternative in glaucoma surgery, but may be less successful in eyes with high risk for failure.