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PURPOSE Subconjunctival mitomycin C has been used in glaucoma filtration surgery with success. A prospective, randomized, masked, placebo-controlled study was performed to evaluate whether single transconjunctival mitomycin C applied either preoperatively or postoperatively would enhance the success of filtration surgery in rabbits. METHODS Two groups of 5 rabbits were studied. In Group I, a Weck-Cel sponge soaked in 0.5 mg/ml mitomycin C was applied transconjunctively for 7 minutes immediately before a full thickness filtering procedure. The other eye was treated similarly with a sponge soaked in balanced salt solution. Group II first underwent filtration surgery followed by treatment with either mitomycin-c or balanced salt solution 3 days later. Postoperative intraocular pressure, bleb status, and complications were evaluated. Treatment failure was defined as postoperative pressure within 4 mmHg of that determined preoperatively or the absence of bleb formation. RESULTS In Group I, mean time to failure (+/- SD) was significantly longer (p = 0.03) in experimental eyes (30 +/- 15.1 days) than control eyes (8.6 +/- 0.8 days). In Group II, the time to failure was 12.4 (+/- 2.6) days versus 9.6 (+/- 2.5) days in the experimental and control eyes respectively, but this was not statistically significant (p = 0.10). Transient limbal vascularization and corneal haze were seen in all experimental eyes. Serious complications included late bleb rupture in eyes pretreated with mitomycin C (all eyes in Group I) and corneal decompensation (one mitomycin-c eye). CONCLUSIONS This study demonstrates that a single preoperative tranconjunctival application of mitomycin-c is more effective at the time of surgery than an application applied in the intermediate postoperative period. Additional studies are needed, however, to further refine both the dose and timing of mitomycin-c application during filtration surgery.