The objective of this study was to determine whether the corneal penetration of 5-fluorouracil (5-FU) could be altered by prodrugs. The prodrugs studied included various 1-alkoxy-carbonyl 5-FU derivatives as well as an 1-acyl-oxymethyl and an 3-acyl derivative. Corneal penetration of 5-FU and its prodrugs was evaluated over 180 min using the isolated albino rabbit cornea in the modified Ussing chamber. 5-FU and its prodrugs were analyzed by reversed phase HPLC. Corneal penetration of 5-FU was very poor due to its low lipophilicity and extensive metabolism during penetration. There was no conclusive evidence for carrier-mediated transport. The corneal epithelium offered the main diffusional and metabolic resistance. Prodrugs of 5-FU with improved lipophilicity dramatically increased the corneal penetration of 5-FU, suggesting that a much reduced dose of 5-FU could be used if corneal penetration and aqueous levels are necessary. It is anticipated that a similar lower dose of prodrug would be required should the prodrug also improve the diffusion of 5-FU across the sclera following subconjunctival injection. Whether dose reduction will lead to reduced corneal toxicity in glaucoma filtration surgery following topical or subconjunctival dosing is an interesting therapeutic opportunity that remains to be determined. In addition to enhancing 5-FU penetration, prodrugs also protected 5-FU from metabolism even though they were rapidly and quantitatively converted to 5-FU during penetration. Protection of 5-FU from metabolism in the cornea may be advantageous from the standpoint of sparing the cornea from toxicity caused by 5-FU, be it from topical or subconjunctival dosing.