Purpose: To establish the effects of single intraoperative exposures to either 5-fluorouracil (5-FU) or two different concentrations of mitomycin C (MMC) on filtration surgery in the rabbit. Methods: A prospective, randomized, masked-observer, placebo-controlled study was performed to compare the effects on filtration surgery in rabbits of a single 5-minute intraoperative exposure to 5-FU (50 mg/ml), MMC (0.2 mg/ml), or MMC (0.4 mg/ml) with control eyes treated with distilled water. Results: Intraocular pressures (IOPs) remained lower for longer and bleb survival was increased (P < 0.05) with all three treatments compared with control eyes. The effect of 5-FU was more transient than MMC. Bleb survival was prolonged in the following order: MMC 0.4 > MMC 0.2 > 5-FU 50 mg/ml. At 30 days, blebs were present in 100% of eyes treated with MMC 0.4 mg/ml, 60% of eyes treated with MMC 0.2 mg/ml, and 0% of eyes treated with 5-FU 50 mg/ml or distilled water. The blebs in the eyes treated with MMC were thinner, and significant complications (endophthalmitis, transient corneal opacification and neovascularization, and a presumed bleb leak) only occurred in the eyes treated with MMC 0.4 mg/ml. Conclusions: The authors' results suggest that 5-FU and MMC at these concentrations, delivered as a single intraoperative dose, prolong the survival of filtration surgery for different lengths of time in the rabbit, which is a model that normally exhibits aggressive healing and rapid failure of filtration surgery. The implications of these potentially titratable effects for filtration surgery in different categories of patients are discussed.