To examine the relevance of concentration of benzalkonium chloride (BAK) on the cornea, we investigated the effects of latanoprost containing BAK alone and in combination with other antiglaucoma drug classes on corneal epithelium in vitro in a cultured rabbit corneal cell line (SIRC) and in vivo, using a corneal resistance device (CRD). [In vitro] staten's seruminstitut rabbit corneal cells were exposed to 0.005% latanoprost for 30 s, followed by either phosphate buffered saline (control), 0.1% brimonidine, 0.5% timolol, 1% dorzolamide, or 1% brinzolamide. The number of viable cells was counted at 8, 15, and 30 min. [In vivo] Albino rabbits were administered one drop of 0.005% latanoprost, followed 5 min later by one drop of an agent from the in vitro trial. This was repeated every 15 min for a total of three times. The change in corneal barrier function was assessed by measuring the corneal resistance at 2 and 30 min after the final administration. [In vitro] At 8 min, the viable cell count in the latanoprost+dorzolamide group was significantly lower than in the control group. At 15 and 30 min, all treatment groups, except the latanoprost+brimonidine group, demonstrated significantly lower viable cell counts than the control group. [In vivo] At 2 min after the final eye drop, the latanoprost+timolol group and the latanoprost+brinzolamide group demonstrated significantly lower corneal resistance than did the latanoprost+brimonidine group. No significant difference was observed between the agents at 30 min. In conclusion, when combining latanoprost containing benzalkonium chloride with other classes of antiglaucoma drugs, brimonidine may cause the least corneal damage, and the number of drug administrations may be an important factor.