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Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits.Primary cultured RCrECL were grown at an air-interface on Clear–Snapwells™ precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na+ and Cl− transport to overall short-circuit current (Isc) was evaluated by removing Na+ and Cl−, respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na+ and Cl− using 22Na and 36Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na+-channel activities to net Na+ fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl2 and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl−-channel, and basolateral K+-channel and Na+(K+)2Cl−-cotransporter activities, respectively, in active ion transport across RCrECL.Isc of RCrECL derived from pigmented rabbits was comprised of 64±2% and 44±5% for active Na+ and Cl− transport, respectively, consistent with net Na+ absorption and Cl− secretion of 0.062±0.006 and 0.046±0.008 μEq/cm2/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited Isc by up to ∼50% with an IC50 of 1 and 0.1 μm, respectively, consistent with participation of apical epithelial Na+-channels to net Na+ absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl2 to the basolateral and bumetanide to basolateral fluid decreased Isc by 86±1.5%, 53±3%, 18±1.8% and 13±1.9% in RCrECL cultured from pigmented rabbits, while 85±0.7%, 36±1.6%, 38±1.8% and 15±3.5% decrea ses are observed for RCrECL from albino rabbits, respectively.Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.