In this study, we have examined the influence of diethylstilbestrol (DES) and 17 beta-estradiol on the proliferation of TM3 Leydig cells, a normalized mouse cell line. Cells were treated with seven different concentrations (1 pg-1 microg/ml) of DES or 17 beta-estradiol, and cell growth was measured at 24-, 48-, and 72-h periods. DES treatment resulted in a significant (p<0.05) stimulation of cell proliferation. We observed two independent peaks of cell proliferation, one at 1 pg/ml DES (186.87%) and the other at 100 ng/ml DES (248.23%). Cytotoxicity was noted at all time periods with 1 microg/ml DES treatment. 17 beta-estradiol treatment resulted in a significant stimulation of cell proliferation (p<0.05) with a trend similar in response to that of DES treatment, as peak proliferation was noted with 1 pg/ml 17 beta-estradiol (125.27%) and 10 ng/ml 17 beta-estradiol (138.31%). Based on these data, it appears that DES is more mitogenic in these Leydig cells compared to 17 beta-estradiol. Furthermore, for the first time, we detected that both DES and 17 beta-estradiol were able to stimulate proliferation of Leydig cells in a biphasic fashion. Cell cycle kinetic analysis revealed that cell entry into the S-phase was higher in the DES treated cells compared to the controls, and doubling times of DES exposed cells were significantly reduced (p<0.05). Co-administration of tamoxifen at a concentration 1000-fold higher than either DES or 17 beta-estradiol resulted in complete inhibition of cell proliferation. Analysis of expression of ER alpha and ER beta by RT-PCR in untreated Leydig cells, as well as Leydig cells exposed to 1 pg/ml DES, revealed that the transcripts of ER alpha and ER beta were not detectable even after 40 cycles of amplification. A 100-ng/ml dose of DES induced ER alpha expression by 20-fold. These data suggest that estrogen exposure-mediated increases in cell proliferation, coupled with the decrease in cell cycle time, may allow greater accumulation of DNA damage to occur in the testicular target cells compared to untreated cells under normal cell cycle control. In addition, an unidentified estrogen receptor may be responsible for the mitogenic activity of estrogens at low levels.