Rabbit (dark irides and albino) and human corneas were excised and mounted in a Lucite chamber in order to determine the permeability of cyclophosphamide-14C and to identify its metabolites. The chromatographic scans obtained from the developed thin-layer plates showed only one peak for either rabbit or human corneas which corresponded to cyclophosphamide. By measuring the permeability of cyclophosphamide across the intact cornea, stroma alone, epithelium/stroma, and stroma/endothelium, it was possible to determine the resistance to penetration for each layer. The epithelium was the rate determining barrier, whereas both the stroma and endothelium offered little resistance. The stroma behaves as an inert sieve with drug diffusing through an aqueous media of gel-like mucopolysaccharide interspersed by a matrix of collagen fibrils.