The mechanisms of estrogenic endocrine disruption on the male reproductive tract are poorly understood. In order to examine estrogenic properties of xenobiotic chemicals on male tissues, we have developed a mouse Leydig cell line (TM3-SF) that self-proliferates under serum-free conditions. This cell line was derived from ATCC's cell line, TM3. The development of TM3-SF was accomplished over a 4-month period by a progressive serum starvation of the original TM3 cells. The newly established cell line was maintained under serum-free conditions for 20 passages prior to testing. Sensitivity of the TM3-SF cells to estrogens was assayed by cell proliferation studies. A total of four compounds, diethylstilbestrol (DES), 17beta-estradiol, 17alpha-estradiol, and Bis-phenol A, were tested. Significant increases in cell proliferation occurred at various concentrations ranging from 1 pg/ml to 100 ng/ml for all four compounds. The order of potency observed was DES > Bis-phenol A > 17beta-estradiol and > 17alpha-estradiol. In addition, we investigated the mechanism for the self-proliferative properties of TM3-SF. The results of these trials indicate that either inhibin or activin is a primary growth factor for this cell line as a 50% inhibition of growth was noted when cell cultures were exposed to the anti-betaa subunit of inhibin/activin. Furthermore, the addition of the anti-betaa subunit of inhibin/activin blocked the DES-induced proliferation of TM3-SF. We conclude that the growth of TM3-SF cells is estrogen sensitive and that either inhibin or activin is involved in the self-regulation of growth.