Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.