Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.