Incubation of dispersed cells derived from an estrogen-responsive mouse Leydig cell tumor with [3H]estradiol in the presence or absence of unlabeled estradiol showed the existence of two types of binding components with high (Kd approximately 10(-9) M) and low (Kd approximately 1.5 X 10(-8) M) affinity respectively. The use of unlabeled diethylstilbestrol as a competitor, however, abolished the low-affinity binder, resulting in the demonstration of the high binding site. This diethylstilbestrol-suppressible binding site was exclusively located in the nuclear fraction, even without hormonal stimuli. A long (5-hr) exposure of these cells to estrogen caused the decrease in the number of nuclear estrogen binding sites, which was similar to so-called 'processing' of putative nuclear estrogen receptor. The direct stimulative effect of estrogen on the proliferation of these cells was demonstrated by measuring cell numbers as well as thymidine incorporation into DNA in the cultured condition. These results would indicate that estrogen directly exerts its effects on this malignant Leydig cell through this unique nuclear binding site.