In these experiments we characterize rabbit and bovine corneal endothelia cell cultured on microporous membrane filters (0.6cm2). Cell cultured bovine or rabbit corneal endothelial cells (subcultures 1–3) were seeded onto Millicell-HA filter inserts. Electrical resistance measured across the cultured monolayers increased steadily through 14 days of culture, reaching 34.2±0.8 ohm-cm (mean±SE) for rabbit cells and 33.1±1.1 ohm-cm2for bovine cells. Alizarin red staining of the monolayers showed a polygonal morphology comparable to that observedin situ.Transmission electron microscopy showed well developed apical junctional complexes and flaps. Exposure of the monolayers to calcium-free medium resulted in the disruption of intercellular junctions, rounding-up of the cells and a decrease in electrical resistance (to near 0). Transmonolayer fluxes of inulin and dextran correlated well to the resistance measurements. Results of this study demonstrate that corneal endothelium, both bovine and rabbit, grown on filter inserts is comparable in morphology and ultrastructure to corneal endotheliumin situ.The cells cultured in this system form functional apical junctional complexes that effect a barrier function comparable to that of the endothelium in situ.