This study demonstrates for the first time, that medroxyprogesterone acetate (MPA) inhibits the proliferation of the estrogen and progesterone receptor negative mammary cancer cell line MFM-223 via the androgen receptor. MPA is a progestin, which is used in the hormonal treatment of disseminated breast cancer. It binds to the progesterone, androgen, and glucocorticoid receptor and may exert its antiproliferative effects via different receptors. MFM-223 human mammary cancer cells contain a very high level of androgen receptors (160 fmol/mg protein) and low levels of estrogen, progesterone, and glucocorticoid receptors (< 20 fmol/mg protein). This cell line provides therefore a good model system to analyze the possible role of the androgen receptor in the action of MPA avoiding interference with other steroid hormone receptors. Effective inhibition of proliferation is achieved by 10 nM MPA or 1 nM of the androgen dihydrotestosterone, corresponding well to the binding affinities of both compounds (3.6 and 0.18 nM, respectively). The involvement of the androgen receptor was confirmed by competition experiments with antiandrogens. Furthermore, MFM-DHT cells, which are an androgen resistant subline of MFM-223 cells, are also resistant to MPA. This data supports the involvement of the androgen receptor in the action of MPA and additionally rules out direct hormone-independent cytotoxic effects of MPA.