A spontaneously established cell line of human retinal pigment epithelium (RPE) was investigated morphologically and studied by chromosomal analysis. Even after the 100th passage, cultured cells maintained epithelioid morphology, and had numerous microvilli and membrane-bound small dense bodies which looked like lysosomes. Abundant rough-surfaced endoplasmic reticulum and mitochondria were observed in the cytoplasm, suggesting that these cells were metabolically active. The absence of pigmentation was noted from the 6th passage. In chromosomal analysis, a small number of 2nd passage cells showed karyotypic changes. At the 12th passage, a single cell line (44, 6-monosomy and Y-missing) was established. By the 100th passage, further karyotypic changes were noted (low tetraploid). The 6-monosomy might have played an important role in the establishment of this cell line. These results indicated that this cell line underwent some changes in chromosomal count and pigmentation but retained many original morphological characteristics. After further evaluation of its characteristics, this cell line, K-1034, could be useful experimental material in the study of the function of the human RPE.