Using high-performance liquid chromatography, we measured Mitomycin C (MMC) concentrations in conjunctiva, sclera and aqueous of 22 rabbit eyes after single topical administration of 0.2 mg.ml-1 MMC during glaucoma filtration surgery. The peaks of MMC concentrations in conjunctiva, sclera, aqueous were 2.01 micrograms.g-1, 2.95 micrograms.g-1 and 0.16 microgram.ml-1 with half life of 0.63, 0.35 and 0.84 hours respectively. Irrigating the ocular surface with 20 ml of normal saline after MMC application reduced the peak drug concentration to 1/8 in conjunctiva, to 1/5 in sclera and to 1/3 in aqueous. The results showed that the MMC concentrations in conjunctiva and sclera were well above the ID50 of rabbit subconjunctival fibroblast (0.1 microgram.ml-1), and the concentration in aqueous was well below the level known to cause endothelium toxicity (approximately 0.2 mg.ml-1) and retinal toxicity (> 1.3 micrograms.ml-1). Therefore, 0.2 mg.ml-1 MMC can inhibit subconjunctival fibroblast effectively and has no side effect on visual function.