We have recently observed that androgens prevent the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PRc) in MCF-7 human breast cancer cells and now report the results of studies that further characterize this new example of sex steroid antagonism. Using a single saturating dose assay to monitor changes in MCF-7 PRc concentration, we have observed that androgens are capable of inhibiting both the estrogenic induction and the ongoing stimulation of PRc synthesis, but have no apparent effect upon basal concentrations of this receptor. Both testosterone and dihydrotestosterone (DHT) demonstrate similar degrees of antiestrogenic activity at concentrations between 10(-10)--10(-8) M. Furthermore, a 10(-8)-M concentration of either androgen completely inhibits the stimulation of PRc synthesis by 10(-11)--10(-8) M 17 beta-estradiol (E2). This inhibitory effect is maintained during the continued presence of either testosterone or DHT, but rapidly disappears after the withdrawal of androgen from the culture medium. The specific nuclear binding of 17 beta-[3H]estradiol over time appears to be similar in cultures incubated in the presence and absence of 10(-8) M DHT. This observation suggests that androgens do not inhibit estrogen action by interfering with the formation, activation, nuclear binding, or nuclear processing of estrogen-receptor complexes. The estrogenic stimulation of PRc is not diminished by the 5 beta-epimer of DHT, and the inhibitory activity of DHT itself is blocked by several different antiandrogens. These findings provide substantial support for the concept that the antiestrogenic effect of androgens is mediated by an androgen receptor mechanism. These results may provide new insights into the clinically apparent antagonistic effects of estrogens and androgens upon both normal and malignant human breast tissues.