The ultrastructure of the round window membrane of humans, monkeys, felines, and rodents discloses three basic layers: an outer epithelium, a middle core of connective tissue, and an inner epithelium. Interspecies variations are mainly in terms of thickness, being thinnest in rodents and thicker in humans. Morphologic evidence suggests that the layers of the round window participate in absorption and secretion of substances to and from the inner ear, and that the entire membrane could play a role in the defense system of the ear. Different substances, including antibiotics, local anesthetics, and tracers such as cationic ferritin, horseradish peroxidase, and 1 mu latex microspheres, are placed in the middle ear side traverse the membrane. Cationic ferritin and 1 micron microspheres placed in perilymph become incorporated by the inner epithelial cells of the membrane. Permeability is selective; factors include size, concentration, liposolubility, electrical charge, and thickness of the membrane. Passage of substances through the round window membrane is by different pathways, the nature of which is seemingly decided at the outer epithelium of the round window membrane.