Glucocorticoid hormone treatment of GR cells, a cultured line derived from mouse mammary tumor tissue, selectively stimulates the rate of transcription of integrated proviral genes specifying mammary tumor virus (MTV). We have incubated isolated nuclei from these cells under conditions in which all three endogenous RNA polymerases appear to be active. RNA synthesized in vitro is distinguished from preexisting nuclear RNA by labeling the in vitro products with [3H]CTP, and the level of MTV RNA synthesis is measured by molecular hybridization with unlabeled viral DNA. Synthesis requires the addition of nucleoside triphosphates, and is inhibited by actinomycin D. Pretreatment of GR cells with dexamethasone, a synthetic glucocorticoid, has no significant effect on the amount of total RNA synthesis in isolated nuclei. In contrast, synthesis of MTR RNA is stimulated 10-20-fold in nuclei from dexamethasone-treated cells relative to untreated control nuclei; the sensitivity of in vitro viral RNA synthesis to inhibition by alpha-amanitin suggests that it is carried out exclusively by RNA polymerase II. The fraction of total RNA synthesis which is viral specific (about 0.2-0.4% in nuclei from dexamethasone-treated cells and 0.01-0.03% in controls) is similar to that detected in pulse labeled RNA from whole GR cells in culture. Our procedures for labeling and hybridization of RNA appear to avoid artifacts recently noted in other in vitro transcription systems.