Human retinal pigmented epithelial (RPE) cells cultured on plastic, unlike RPE in situ, often fail to density arrest, do not produce melanin, and do not express mRNA for cellular retinaldehyde binding protein (CRALBP). When human RPE are cultured on the extracellular matrix produced by bovine corneal endothelial cells for 1 week or human RPE for 3 weeks, they density arrest, assume a differentiated morphology, and produce pigment and mRNA for CRALBP (all differentiated features of RPE). Human RPE grown on a laminin substratum or grown in media supplemented with basic fibroblast growth factor (basic FGF), assume a differentiated morphology for up to 3 weeks, and this is maintained for several months when the cells are grown on laminin in the presence of basic FGF and heparin. With the latter conditions, the cells also produce mRNA for CRALBP. Human RPE grown on bovine corneal endothelial cell matrix treated with neutralizing antibodies to basic FGF or laminin or agents that displace FGF from extracellular matrix (suramin or protamine), do not assume a differentiated morphology and express less CRALBP mRNA than RPE grown on bovine corneal endothelial cell matrix treated with antibodies to type IV collagen. These data suggest that the extracellular matrix may facilitate RPE expression of differentiated features and that laminin and basic FGF may be important components.