The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the Dl, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the Dl or D2 HAN outgrowth line, in the CD8 DH outgrowth line, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormoneor 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice. Murine mammary tumorigenesis is a multistage process that is influenced by several factors, including the mouse mammary tumor virus (MuMTV), hormones, and carcinogens (1, 2,22). BALB/c mice, which have a low incidence of spontaneous mammary tumors (22), contain five to nine copies of the endogenous, germinally transmitted MuMTV per diploid cell (1, 21). MuMTV RNA was low or undetectable in BALB/c mammary glands from breeding females and in spontaneous mammary tumors (14, 26, 27). In contrast, glands and tumors from the high-tumor-incidence strain BALB/cfC3H, which contain an exogenous milk-transmitted virus designated MuMTV-S (1), had high amounts of MuMTV RNA (26, 27).