RNA synthesis catalyzed by nuclei isolated from mammary glands of pregnant and lactating rats and from 7,12-dimethylbenz(α)anthracene-induced mammary tumors was examined, and the following observations were made.(1) Assay 1 (6 mM Mg2+, low ionic strength, nucleolar polymerase I), and assay 3 (2 mM Mg2+, 1.6 mM Mn2+, 0.3 M ammonium sulfate, high ionic strength, nucleoplasmic polymerase II) promoted synthesis of ribosomal-like (rRNA) and nonribosomal-like RNA (dRNA), respectively, as verified by differential sensitivity to actinomycin D, double-labelling experiments, and response to α-amanitin.(2) Synthesis of a rRNA-like product was increased in assay 2 (6 mM Mg2+, 0.02 M ammonium sulfate), compared with assay 1; in assay 3 increased formation of a more dRNA-like product occurred. Stimulation of mammary gland nuclear RNA synthesis by ammonium sulfate is biphasic, similar to the response of rat liver and sea urchin nuclei.(3) In assay 1, more rRNA was synthesized at 30° than at 37°; in assay 3 somewhat greater synthesis of dRNA occurred at 37° compared with 30°. The pH optima in assays 1 and 3 of 8.0 and 8.5, respectively, are the opposite of those reported for rat liver nuclei.(4) During pregnancy, isolated nuclei synthesized more dRNA-like product, compared to lactation; nuclei from lactating glands formed more rRNA, than during pregnancy. Nuclei from slowly growing tumors (Ts) examined in the three assays were 30–50% as active, while those from rapidly growing tumors (Tf) exceeded the activities of nuclei from pregnant (P) or lactating (L) rats.(5) P, L, and Ts nuclei incubated in assay 3 with α-amanitin were inhibited about 60%, compared to an 80% reduction with nuclei from tumors that were growing rapidly. The ratio of the base G to either A or U did not return to that of assay 1 or 2 (rRNA). This result is consistent with the presence of a third nucleoplasmic RNA polymerase (enzyme "III"), as described with rat liver and sea urchin nuclei.(6) Nuclei from proliferating normal and neoplastic tissues exhibited greater polymerase II activity, and the ratio of nucleoplasmic to nucleolar enzyme activity was increased. Changes in activities of enzyme II and enzyme "III" do not appear to be necessarily coordinate.(7) The pattern of polymerase activity in P and L nuclei is probably related to cellular proliferation and/or hypertrophy during pregnancy, and cellular function represented by milk protein synthesis with lactation. RNA polymerase activity in T nuclei correlated with the growth rate of the parent tumor and not with its histology, at least not in any simple way.