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DNA-dependent RNA polymerases were extracted from rat uterine tissue, partially purified and resolved by DEAE-Sephadex chromatography. RNA polymerases I, II, IIIA, and IIIB eluted at the characteristic ammonium sulfate concentrations of 0.15, 0.28, 0.34, and 0.42 M, respectively. The sensitivity of each peak of polymerase to alpha-amanitin was examined and was shown to be essentially identical to the three classes of RNA polymerases in other mammalian systems. RNA polymerase I was insensitive to high levels of alpha-amanitin, RNA polymerase II was sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.006 mug/ml) and RNA polymerases IIIA and IIIB were sensitive to high concentrations of alpha-amanitin (50% inhibition at 18 mug/ml). The alpha-amanitin sensitivity curve of total RNA synthesis measured in isolated nucleo demonstrated that the activity of each class of RNA polymerase could be quantitated in uterine nuclei. Thus the initial decrease in activity at low concentrations of alpha-amanitin (50% inhibition at 0.005 mug/ml) was attributed to the inhibition of RNA polymerase II activity, the second decrease in activity at higher concentrations of alpha-amanitin (50% inhibition at 15 mug/ml) was attributed to the inhibition of RNA polymerase III activity, and the activity which was resistant to the highest alpha-amanitin concentration tested was attributed to RNA polymerase I activity. When estradiol was given to immature rats 6 h before killing both RNA polymerases I and III levels in nuclei were increased significantly over the control values. The time course of these changes demonstrated that the increases in RNA polymerases I and III were first evident between 1.5 and 3 h following hormone treatment. Significantly, these increases in polymerase I and III in nuclei parallel the published increases for rRNA and tRNA synthesis following hormone treatment. However, the amount of RNA polymerase I and III was not altered upon extraction, suggesting that these changes are due to the alteration in chromatin template activity. Both estradiol and estriol produced identical increases in uterine RNA polymerase I and III 6 h after treatment.