The purpose of this study is to characterize transport of acebutolol through the corneal epithelium. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate the drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL^® insert coated with fibronectin and were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Measurements of acebutolol permeability through RCEC layer were carried out to assess transcellular permeability coefficient (P_transcell) in the absence or presence of inhibitors. Paracellular permeability coefficient (P_paracell) was calculated by permeability coefficient of hydrophilic drugs (P_cell). The transcellular permeability of acebutolol from apical side to basal side (A-to-B) showed concentration-dependency. The acebutolol flux in the A-to-B direction was smaller than that of opposite direction. Sodium azide, verapamil, and cyclosporin A enhanced the transcellular permeability of acebutolol in the A-to-B direction. Acebutolol permeability through an excised rabbit cornea was also increased by verapamil. Thus, it was suggested that acebutolol was actively secreted via P-glycoprotein in a corneal epithelium.