The success rate of filtration surgery has been increased by the postoperative subconjunctival injection of 5-fluorouracil, a potent antimetabolite. However, the optimal route of administration has not been determined. Trabeculectomy was performed on one eye each of 14 patients. Topical 5-fluorouracil was applied intraoperatively (50 mg/ml for five minutes) and subconjunctival 5-fluorouracil was injected postoperatively (an average of 5.8 injections) (mean total dose, 29 mg). Seven of the 14 eyes had primary open-angle glaucoma, and seven eyes had open-angle glaucoma with either uveitis, aphakia, or previous failed trabeculectomy. Mean preoperative intraocular pressure was 24.7 mm Hg during treatment with an average of three antiglaucoma medications, and mean final intraocular pressure was 11.9 mm Hg during treatment with an average of 0.2 medication. Thirteen of 14 eyes (93%) had final intraocular pressure of 18 mm Hg or less. Mean follow-up was 6.4 months (range, four to nine months). No remarkable complications occurred. Visual acuity remained stable in 13 of 14 eyes (93%). Intraoperative 5-fluorouracil may be a helpful adjunct in achieving low final intraocular pressure after trabeculectomy.