Hormonal regulation of mammary tumor virus (MTV) production, has been analyzed with normal mammary epithelial cells from chronically infected BALB/cfC3H mice. The effect of prolactin in terms of increased MTV production was not reproducibly observed in cells cultured in tissue culture dishes, whereas the cells grown on floating collagen gels consistently responded to prolactin in a dose-dependent manner. Of the three media tested. Dulbecco's modified Eagle's medium was found to be the best in terms of responsiveness to prolactin and in maximal MTV production. Specificity studies with other pituitary and placental hormones in place of prolactin have shown that both growth hormones and human placental lactogen can replace prolactin, whereas follicle-stimulating hormone, luteinizing hormone, and thyrotrophin were ineffective. Contrary to the mammary tumor systems, where it has been shown that insulin and glucocorticoid can maximally stimulate MTV production, these hormones alone elicited only a small response in the absence of prolactin in normal mammary epithelial cells. Although prolactin alone had very little effect by itself, its presence was necessary (permissive effect) in order for the glucocorticoids to be able to maximally stimulate MTV production in normal cells.