Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.