The cone matrix sheath is a biochemically and structurally distinct domain of the retinal interphotoreceptor matrix that is specifically associated with cone photoreceptor cell inner and outer segments. An aqueous extraction technique for the isolation of cone matrix sheath-enriched retinal fractions was developed and used to examine the structure and composition of this extracellular matrix domain. A complex substructural organization of the cone matrix sheath was observed. Many longitudinally oriented, filamentous structures extend the entire length of the sheath and terminate in filamentous rings at both the apical and basal ends. These longitudinal filaments are interconnected by a finer, anastomosing filamentous network. The basal rings of cone matrix sheaths are interconnected with similar rings of matrix material associated with rod photoreceptor inner segments. Gel electrophoresis and lectin blot analyses of cone matrix sheath-enriched fractions reveal the presence of 17- and 32-kilodalton bands, labeled by peanut lectin, that are likely to be components of the structural elements of cone matrix sheaths. Thus, structural elements potentially capable of mediating adhesion between the neural retina and the retinal pigmented epithelium are present in the cone matrix sheath and may be at least partially responsible for the observation that cone matrix sheaths are firmly adherent to the pigmented epithelium and the neural retina.