SummaryIncubation of estradiol in vitro at 25° C with homogenates of carcinogen-induced mammary tumors of ovariectomized rats stimulated the magnesium-dependent RNA polymerase activity of nuclei of the hormone-dependent (HD) (regressing) tumors, but had no effect on this activity in nuclei of hormone-independent (HI) (growing) tumors. Furthermore, recombination of the nuclei and cytosol fractions of HD and HI tumors indicated that the in vitro effect of estradiol on subsequent tumor nuclear RNA synthesis required the estrogen receptor-containing cytosol but was specific to nuclei of the HD tumor. This constituted the first direct in vitro effect of estrogen on a specific biochemical process in an HD mammary tumor.