An in vitro cell-free system of uterine nuclei and cytosol receptors has been used to analyze the effects of aging on estrogen stimulation of RNA polymerase II activity. By using fixed concentrations of nuclei and cytoplasmic receptor--estrogen complexes (R-E2), it was found that mature nuclei are 3 times more efficient (155% vs. 57%) than old ones for stimulation of polymerase activity by mature R-E2. Meanwhile, mature R-E2 are 5 times more efficient (155% vs. 31%) than old ones in supporting such stimulation in mature nuclei. Stimulation by old cytosol R-E2 is so poor that it is essentially unaffected by nuclear age (31% with both mature and old nuclei). Finally, equimolar mixtures of mature and old cytosol R-E2 stimulate polymerase II activity in mature nuclei by 77%, a value intermediate between mature and old cytosols used separately. These results indicate that both nuclei and cytosol from old uteri are deficient in their ability to support estrogenic stimulation of RNA polymerase II.