PURPOSE: The retinal pigment epithelium (RPE) in vivo is known to have polarized membrane domains that are essential for its normal function. Unless the proper cell culture conditions are used, these polarized features are often lost. In the past, the use of Chee's Essential Medium (CEM) in our RPE cultures has produced functional polarity of the cell monolayers. Unfortunately, except by custom formulation, which is costly, this product is no longer commercially available. We therefore sought to develop a replacement culture medium that would support morphological and functional polarity of RPE membrane domains when the cells are removed from the in vivo milieu. METHODS: To test the performance of this CEM replacement medium in comparison with three other culture media, we grew fetal human RPE to confluence on Millipore Millicell culture wells. We then used Na,K ATPase as a membrane domain marker by displaying it with polyclonal antibodies. This marker was chosen because it is not always properly polarized in culture. Immunofluorescence was imaged by laser confocal microscopy of whole mounted intact monolayers on their Millicell supports. We also used transepithelial resistance (TER) as a measurement of functional polarity as well as bestrophin protein expression as an index of cell differentiation. The expression of Na,K ATPase and bestrophin was confirmed by Western blot analysis of whole RPE cell extracts. RESULTS: Immunofluorescence labeling of cultured RPE Na,K ATPase was observed exclusively on the apical membrane when the CEM replacement or DMEM with high glucose was used. However Na,K ATPase was not completely polarized in DMEM/F12 medium and the cells did not express detectable Na,K ATPase in DMEM with low glucose. Western blots showed that Na,K ATPase was expressed at similar levels in CEM replacement, DMEM with high glucose and DMEM/F12 as indicated by the intensity of an approximately 100 kDa band representing the a subunit. The CEM replacement gave superior TERs as well, ranging from about 2 to 5.6 fold higher than the other media. Bestrophin protein was readily detectable by Western blot in CEM replacement medium whereas it was barely detectable in DMEM/F12 and undetectable in DMEM with high and low glucose. CONCLUSIONS: We have provided immunocytochemical evidence that the CEM replacement medium supports the appropriate membrane domain expression of Na,K ATPase when the cells are grown on Millicell chambers. Excellent TERs and robust expression of bestrophin are also observed. This combination of features was not observed when other, standard culture media were used. The results suggest that, under these conditions, cultured human RPE develops a highly differentiated and functional polarity appropriate for the in vitro modeling of RPE in vivo function.