Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and

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... lin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0\m=.\75\m=x\106 cells cm\m=-\2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.