The authors measured mitomycin C (MMC) concentrations in ocular tissues with high-performance liquid chromatography. Mitomycin C concentration after a single subconjunctival injection of the drug in rabbit eyes showed a rapid decrease with a half-life of 0.18 to 0.30 hours for the conjunctiva and 0.20 to 0.45 hours for the sclera at the injection site. Irrigating the ocular surface with 200 ml of saline after MMC application reduced the initial drug concentration to one fifth in the sclera and to one fifteenth in the conjunctiva but did not change the half-life. The MMC concentration in human trabeculectomy specimens obtained immediately after MMC application (0.2 mg/0.5 ml) and irrigation was 5.4 to 12.0 micrograms/g with a mean of 8.4 micrograms/g, a level similar to that in the rabbit sclera immediately after the irrigation after administration of the same MMC dose. These results indicate that MMC disappears rapidly from the ocular tissues and that irrigating the tissues significantly reduces the tissue concentration of MMC.