Summary. Epididymal epithelial fragments, free of stromal elements were isolated from mature rats using two sequential collagenase digestions. Within 24 h these attached efficiently to a variety of substrates including glass, plastic, placental collagen, type IV collagen and epididymal extracellular matrix material. Cells spreading away from the fragments rapidly assumed a flattened, overlapping, monolayer appearance typical of epithelial cells in culture. Cells still associated with the fragments or adjacent to them remained more polarized and more closely resembled epididymal principal cells in vivo than did cells that had migrated to the periphery of the monolayer. Apical microvilli characteristic of these cells in vivo were common during the first 4 days in culture but diminished in number and size thereafter. Cultured cells maintained many of the structural features characteristic of principal cells in vivo, including a well developed Golgi apparatus, coated pits and vesicles, and many multivesicular bodies. An extensive filamentous network, shown immunocytochemically to consist of keratin, was present in the cytoplasm of all cells but was more obvious in flattened cells at the periphery of the monolayer. Rhodamine phalloidin labelling of filamentous actin showed that concentrations of actin occurred corresponding to microvilli on the apical surface, in a continuous ring just below the apical surface, and also in stress fibres at the base of the cells. Cells isolated and cultured from the distal caput epididymidis possessed lobulated nuclei, in contrast to the round or oval nuclei found in cells cultured from the proximal caput epididymidis. Cells from the distal caput epididymidis were also characterized by the presence of many lipid droplets in their cytoplasm. Autofluorescent granules were observed in epithelial cells from both regions but were larger and more numerous in cells isolated from the distal caput epididymidis. Tritiated thymidine incorporation by the cells after 4 days in culture showed that cells adjacent to the parent epithelial fragment were dividing at a greater rate than cells that had migrated to the periphery of the monolayer.