Cell lines were established from cultures derived from Fischer rat embryos according to the transfer schedule described by Todaro and Green (1963) for mouse 3T3 cells where cell crowding and serum exhaustion were kept to a minimum. Cell growth rate did not decline greatly during the course of successive 3-day transfers. Like 3T3 cells the rat cell lines possess very low saturation desities under standard culture conditions. A clonal cell line with a relatively high plating efficiency as obtained from one of the cell lines, 3YL. In these cloned cultures, virus growth was not detectable upon infection with SV40, while a small amount of virus was produced upon infection with polyoma virus. Morphological transformation of the cloned 3Y1 cells by SV40 and polyoma virus could be assayed with single-hit kinetics and with effieiencies comparable to those of the previously available transformation systems for each virus. Independent cell lines transformed by SV40 were consistently virus-free and all the lines tested produced SV40 upon fusion with permissive monkey cells. Most of the independent transfromed cell lines isolated after polyoma infection appeared to be virus-free, although the cultures of some lines produced a small amount of polyoma virus spontaneously after a prolonged cultivation. Most of the virus-free polyoma-transformed lines produced virus upon fusion with permissive mouse cells.