Autosomal dominant polycystic kidney disease is characterized by the formation of large fluid-filled epithelial cysts. To obtain renal cyst wall epithelium for in vitro study, we employed an explant culture technique using medium-hydrated collagen gel as the culture substrate. Pieces of excised cyst wall were submerged within collagen gel. Cells of the cyst lining migrated to form a polarized epithelium at the surface of the surrounding collagen gel. Regions of the outgrowth were isolated by microdissection and used as a source of cells for subculture. Cyst-derived epithelium retained the ultrastructural features of the renal cyst of origin. The cells were cuboidal and bore short apical microvilli. Cells often were joined by apical tight junctions. Intercellular channels were narrow and bordered by short microvillus projections at the basolateral membrane. Epithelial cells rested on a densely staining basal lamina. The explants commonly developed small solitary cysts within their wall that were filled with fluid. These mural cysts were lined by a simple epithelium morphologically similar to the cells that lined the explant. Explantation of human renal cyst wall to culture within collagen gel provides a reproducible method to isolate autosomal dominant polycystic kidney disease epithelium for subculture. This method offers an alternative to the use of proteolytic enzymes to establish morphologically stable cultures of cyst lining cells for use in experimental renal cystic disease.