Current research and an overall review of 25 years of round window membrane studies are presented. The approach, rationale and concepts that have evolved from these studies are described. Ultrastructural studies of the round window membrane of humans, monkeys, felines and rodents have disclosed 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 thickest in humans. Morphologic evidence suggests that the layers of the round window participate in resorption and secretion of substances to and from the inner ear, and that the membrane could play a role in the defense system of the ear. Different substances, including antibiotics and tracers, when placed in the middle ear side traverse the membrane. Tracers placed in perilymph become incorporated into the membrane by the inner epithelial cells. Permeability is selective and factors affecting permeability include size, concentration, electrical charge, thickness of the membrane and facilitating agents. Passage of substances through the membrane is by different pathways, the nature of which is seemingly decided at the outer epithelium of the membrane. Round window membrane studies have provided increased knowledge of the anatomy and function of this structure, as well as new insights into pathology and pathogenesis. The concepts that have evolved from these studies are potentially useful for understanding middle and inner ear interactions, and for eventual drug delivery (based on permeability) to the inner ear.