The overall objective of this study was to evaluate the effect of chitosan, benzalkonium chloride (BAK) and disodium ethylendiaminetetraacetic acid (EDTA), alone and in combination, on permeation of acyclovir (ACV) across excised rabbit cornea. Corneas of male New Zealand White rabbits were used in these studies. Transcorneal permeation studies were conducted at 34 degrees C using a side-bi-side diffusion apparatus. In the presence of 0.01% BAK, transcorneal permeability of ACV was observed to increase almost 10.5-fold, from 3.5x10(-6) to 37.4x10(-6)cm/s. At 0.005% BAK, permeability of ACV was almost 3-fold higher than control. Combination of BAK 0.005% and EDTA 0.01% increased transcorneal penetration of ACV by 2.5-fold. Chitosan 0.2 and 0.1% increased corneal permeability of ACV by 5.8- and 3.1-fold, respectively, whereas, at 0.02%, chitosan did not exhibit a statistically significant effect. BAK at 0.005%, in combination with 0.01% EDTA and 0.1% chitosan, increased transcorneal ACV permeation by 5.5-fold. This study suggests that a judicious combination of chitosan, BAK and EDTA can lead to a significant increase in ACV's transcorneal permeability and that chitosan can enhance diffusion of hydrophilic agents across the corneal membrane. Further in vivo evaluation is warranted.