Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93,000. These receptors internalize 125I-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100,000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surface receptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures have no apparent association with cytoskeletal elements.