A tissue culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the efferent ductules in boars. A currently used culture protocol for rat epididymal epithelium was used as a starting point and was subsequently modified because of unsatisfactory results. Epithelial plaques were isolated by mechanical uncoiling of the ductules and two sequential enzymatic digestion steps. Plaques were seeded onto extracellular matrix-coated permeable membranes and maintained in a two-chamber system. Samples taken before seeding and after 7 days in culture were processed for transmission and scanning electron microscopy. Perfusion-fixed material from earlier studies served as a reference to assess ultrastructural preservation. In addition, endocytotic activity was investigated by adding cationized ferritin to the culture medium on day 8 before fixation. At the end of the disaggregation procedure, cells were cuboidal, while cilia, microvilli and cell organelles were well preserved. After 7 days in culture, three types of cell formation were observed: cysts, pseudotubules and epithelial sheets. Cell sheets were made up of closely juxtaposed cells bearing motile kinocilia and exhibiting well-developed polar differentiation, as judged from the localization of cell junctions and organelles. Although it did not return to the values of native material, cell height was greater than that of cells grown according to the pre-existing protocol. Furthermore, preferential uptake of ferritin by principal cells after 7 days in culture was demonstrated. Preservation of these fundamental characteristics of the in vivo state corroborates that our in vitro system will furnish reliable information.