The purpose of this study is to characterize the transport of tilisolol and timolol through the corneal epithelium, which is believed to be a tight barrier of ocular drug absorption. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Beta-blocker permeability through the RCEC layer was measured to assess the transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. The transcellular permeability of tilisolol was dependent on drug concentration although timolol showed no concentration dependency. Tilisolol flux from the apical to the basal side was larger than in the opposite direction although timolol showed no direction dependency. The transcellular permeability of tilisolol from the apical to the basal side was inhibited by sodium azide, tetraethylammonium, quinidine, taurocholic acid, guanidine and carnitine. Tilisolol had an active mechanism in uptake to the corneal epithelium, probably by the organic cation transporter family, although timolol predominantly permeated via passive diffusion. This RCEC system was useful to characterize the ocular permeation mechanism of drugs.