Purpose.To investigate the permeation of cyclosporin A (CsA) through fresh and frozen human corneas in the presence and absence of three penetration enhancers: benzalkonium chloride (BZCl), dimethylsulfoxide (DMSO), and Cremophor-EL.Methods.Human corneas, unsuitable for transplantation, were either freshly used for permeability experiments or snap-frozen in liquid nitrogen and stored at −85°C. CsA permeation through either fresh or thawed frozen corneal tissue was determined using a flow-through diffusion apparatus (20°C for 24 hours). Flux rates for CsA were determined in the presence and absence of the penetration enhancers 0.01% BZCl, 20% DMSO, and Cremophor-EL (10% and 20%). Analysis of variance and Duncan's multiple-range test were used to test for steady state, and an unpaired Studentttest with Welch's correction was used to test for differences between the mean flux values at each time point. A significance level of 5% was used for all of the statistical tests.Results.No statistically significant differences in flux values of CsA could be detected between fresh and frozen corneas. In the presence of Cremophor-EL (10% and 20%) and 0.01% BZCl, statistically significant increases in flux values of CsA before 16 hours and after 16 hours, respectively, could be observed. In the presence of 20% DMSO, no statistically significant increases in flux values could be detected.Conclusions.The permeation of CsA through human corneas appeared to be enhanced by the presence of BZCl and Cremophor-EL. Additionally, it was shown that the flux rate of CsA across fresh and frozen corneas was not significantly different.