The interplay of corneal epithelial integrity and prodrug lipophilicity in the corneal penetration of a homologous series of timolol prodrugs was studied. A reversed phase HPLC procedure was used to assay for timolol and its prodrugs. The optimal distribution coefficient for corneal penetration on a logarithmic basis was found to be 2.5, and for two prodrugs of equal lipophilicity the enzymatically labile one was better absorbed. The corneal permeability coefficient of O-acetyl, propionyl, and butyryl timolol was unaffected by deepithelizing the cornea, whereas that of the other prodrugs was reduced. This suggests that these three prodrugs diffused across the corneal epithelium proper as readily as across its basement membrane, which was not removed by the deepithelizing procedure. In contrast to the in vitro results, deepithelizing the cornea did not affect the aqueous humor concentration of timolol at 20 min afforded by any of the prodrugs except the O-propionyl and octanoyl esters. This suggests that factors other than corneal penetration exerted a more important role in affecting the aqueous humor drug concentration when the cornea was deepithelized.