The retinal pigment epithelium (RPE) plays a critical role in the development and maintenance of adjacent photoreceptors in the vertebrate retina. This study describes the development and characterization of ARPE-19, a spontaneously arising human RPE cell line with normal karyology which forms polarized epithelial monolayers on porous filter supports. The cell line was established by selective trypsinization of a primary RPE culture resulting in a uniform population of highly epithelial cells which exhibit a strong growth potential. To determine the extent of biochemical differentiation, the expression of the RPE-specific markers CRALBP and RPE65 was examined by Northern analysis. A single 1.6 kb CRALBP mRNA transcript and a single 2.8 kb RPE65 transcript were detected in samples of ARPE-19 total mRNA. The expression of CRALBP protein in ARPE-19 cell lysate was detected by Western blot analysis and immunocytochemistry was used to detect CRALBP throughout the cytoplasm of most, but not all, cells in confluent cultures. The essential criteria for monolayer formation were determined experimentally and it was found that ARPE-19 cells exhibit morphological polarization when plated on laminin-coated Transwell-COL filters in medium with a low serum content. The time course of tight-junction formation was determined by recording the transepithelial resistance of monolayers and reached a maximum of 50-100 omega cm2 after 4 weeks of culture. Barrier properties of ARPE-19 monolayers were evaluated by measuring the flux of 3H-inulin from the apical to the basolateral compartment of cell culture chambers. Finally, ARPE-19 clonal sublines were generated by serial dilution in an attempt to produce a subline with a high transepithelial resistance (TER). The morphology of the sublines was variable and the cloned cells exhibited a tendency to senesce in culture, confirming that this cell line is not transformed. No subline monolayers developed a TER greater than those recorded for the parent cells. Our results demonstrate that ARPE-19 has structural and functional properties characteristic of RPE cells in vivo and suggest that this cell line will be valuable for in vitro studies of retinal pigment epithelium physiology.