Five milligrams of fluorouracil was injected subconjunctivally in the rabbit eye, and its concentration changes in the cornea, aqueous humor, and conjunctiva and sclera (both at the injection site and 180 degrees away from it) were determined by microbiological assay. The fluorouracil concentrations in the cornea and the aqueous humor averaged about 20 micrograms/g at one hour. The former decreased to 0.5 micrograms/g at 24 hours, while the latter was 0.03 micrograms/g at ten hours or later. At five hours, the fluorouracil concentrations in the conjunctiva and sclera at the injection site were similar to those 180 degrees away from it, both averaging about 2 micrograms/g. They decreased to 0.3 through 0.9 micrograms/g at 24 hours. The fluorouracil concentrations in the cornea, conjunctiva, and sclera at 24 hours were still above the reported 50% inhibition levels for the cultured conjunctival fibroblast. These findings have potential clinical implications for the safe use of fluorouracil.