Transcorneal in vitro permeation studies of ophthalmic drugs are normally performed with either excised animal corneas or latterly corneal cell culture models. A good correlation between these models and excised animal corneas regarding permeation behaviour of drugs has already been shown. However, comparisons between corneal in vitro models containing human cells and excised human corneas do not exist yet. Therefore in the present study the transcorneal permeation of six different model drugs (pilocarpine hydrochloride, befunolol hydrochloride, hydrocortisone, diclofenac sodium, clindamycin hydrochloride and timolol maleate) across our previously described three-dimensional organotypic human cornea construct (HCC) was tested using Franz diffusion cells and compared with permeation data obtained from human donor corneas. The HCC showed a similar permeation behaviour compared with human donor cornea for all substances. The permeabilities (permeation coefficients P) of the human cornea equivalent versus the human donor cornea were the same in the case of diclofenac, clindamycin, timolol, but marginally decreased for hydrocortisone and slightly increased for pilocarpine and befunolol. These small differences of permeation coefficients were expressed as factors and only varied from 0.8 to 1.4. The results indicate that the HCC may be an alternative for in vitro permeation studies and appropriate for predicting drug absorption into the human eye.