A new continuous cell line derived from a human retinoblastoma has been established. This cell line, WERI-RB1, has been maintained in vitro since December 1974. The purpose of this investigation was to characterize WERI-Rb1 on the basis of morphology, growth, tumorigenicity, cytogenetics, and to compare this cell line with Y79, a human retinoblastoma cell line established at another institution. Morphologically, both cell lines were similar; each spontaneously grew as a suspension of small round cells in grapelike clusters. Each exhibited growth of cells in rosettes, as well as unusual chain formations. Growth rates differed: the population-doubling times for WERI-Rb1 and Y79 were 96 and 33 hr, respectively. When the negative surface charge on a plastic tissue culture flask was changed, each cell line grew as a monolayer. Y79 could be cloned in soft agar; WERI-Rb1 could not. An inoculm of 10(7) WERI-Rb1 or Y79 cells produced a retinoblastoma in test rabbits. Karyological examination showed each cell line to have a stable, near diploid chromosome number. Although large markers were observed in each line, they shared no common marker.