The purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctional permeabilities of FITC-dextran 4400 (FD-4) was also determined through the cornea in the presence of ophthalmic ingredients. Intact cornea showed approximately one k-ohm×cm² of transepithelial electrical resistance (TEER) and extremely low permeability of FD-4. The ophthalmic ingredients used in the present study were benzalkonium chloride (BK; 0.002%, 0.01%, 0.05%), ethylenediaminetetraacetic acid (EDTA; 0.5%), capric acid (C10; 0.25%), saponin (SP; 0.1%), taurocholic acid (TA; 1.0%) and sodium dodecyl sulfate (SDS; 0.01%). They were previously reported to be effective on corneal penetrations of various drugs at those concentrations without severe toxicity. These ingredients decreased TEER and increased corneal permeability of FD-4. BK reduced TEER in a concentration-dependent manner. There was a significant correlation (γ=0.860) between the permeability coefficient (Papp) of FD-4 and conductance (Gm), which is the reciprocal value of TEER. It was also indicated that Papp and Gm have a relationship with the corneal cytotoxicity of the ingredients. In conclusion, an electrophysiological method using isolated cornea was very useful to determine the continuous and real-time influence of ophthalmic ingredients on the cornea. In this method, electrophysiological conductance must be able to predict corneal tight junction permeability and the corneal cytotoxicity of ingredients.