To overcome the technical difficulties encountered in investigating the physiology of Reissner's membrane (RM), primary culture of RM was established. Minute explants obtained by mechanical dissociation were seeded on impermeable substrate coated with collagen type I in serum-free, hormone-supplemented medium. Within 2 weeks, a large cultured area was obtained which reproduced the two cell layers of the original explant. One layer, deep or superficial depending on the side to which the explant adhered, was made of cells exhibiting light and electron microscopy characteristics of an epithelium. Immunohistochemical methods revealed that they expressed cytokeratin, demonstrating their epithelial nature. The second layer was made of mesenchymal cells. This study suggests that cell culture of RM will facilitate further investigation of its physiology.