The hormonal regulation of mouse mammary tumor virus (MMTV) RNA in normal mouse mammary epithelium was studied in an explant system. In tissue from parous mice, physiological concentrations of prolactin stimulated MMTV expression, while only pharmacological concentrations of cortisol were effective. Regulation in explants from virgin mice was similar to that in parous animals except that the former were less sensitive to prolactin; this relative unresponsiveness may explain why uninduced tissue from virgin mice does not express MMTV RNA, while that from parous mice does exhibit some basal production. These results suggest that prolactin plays a major role in MMTV expression in normal mammary epithelium and that glucocorticoids may only have a permissive effect or may act through an indirect mechanism requiring high concentrations. These data also suggest that the greater susceptibility of parous mice to MMTV-induced tumorigenesis may reflect the greater prolactin sensitivity in the glands from these animals.