A prospective, randomized, masked, placebo-controlled study was performed to determine whether a single, intraoperative subconjunctival application of a 0.5-mg/mL solution of mitomycin-C enhances the success of full-thickness filtration surgery in rabbits. Compared with control eyes, mitomycin-C-treated eyes showed significant increases in bleb duration from 8.1 +/- 2.4 to 68.0 +/- 20.8 days and in intraocular pressure reduction from 6.0 +/- 3.0 to 63.6 +/- 21.5 days. Histopathologic evaluation confirmed the inhibitory effects of mitomycin-C on fibrovascular, fibrocellular, and collagenous organization of the filtering blebs that resulted in their preservation. Transient, superficial, corneal vascularization anterior to the bleb occurred in all mitomycin-C-treated eyes. No other clinical or pathologic signs of undesirable side effects were noted. We studied the effectiveness and safety of a single intraoperative application of mitomycin-C in prolonging the success of filtration surgery in rabbits and its potential for similar use in humans.