Glucocorticoid hormones specifically increase the intracellular concentration of mouse mammary tumor virus (MMTV) RNA in a cultured cell line from a GR mouse mammary carcinoma (GR) and in an MMTV-infected rat hepatoma cell line (M1.19). In contrast, these steroids have no effect on the concentration of MMTV RNA in a lymphoma line, S49, from a Balb/c mouse. Using a molecular hybridization procedure to detect newly synthesized RNA, we have directly measured the effect of dexamethasone, a synthetic glucocorticoid, on the rate of MMTV RNA synthesis. In GR cells the hormone causes a 10-fold increase in the rate of synthesis of viral RNA without appreciably affecting the overall rate of cellular RNA synthesis. The transition from the basal to the maximally stimulated rate of MMTV RNA synthesis occurs within the earliest labeling period, 0-15 min after addition of the hormone. Thus, it appears that glucocorticoids regulate MMTV genes principally by this rapid and specific alteration of their rate of transcription. Similar results are obtained in M1.19 rat hepatoma cells. In contrast, dexamethasone does not affect the rate of viral RNA synthesis in S49 lymphoma cells.