Nuclear RNA polymerase activity was studied in homotransplanted rat glial tumors where the primary tumor was produced by transplacental injection of ethylnitrosourea. Alpha amanitin, cycloheximide, and rifampicin were tested as inhibitors of this activity. Alpha amanitin significantly inhibited RNA polymerase activity in all tumors. This indicated that the major nuclear RNA polymerase activity seen in vitro in the tumor nuclei was RNA polymerase II. This is similar to the activity seen in normal glial nuclei. Cycloheximide and rifampicin which have no effect on RNA polymerase activity in normal glial nuclei inhibited about 20% of the polymerase activity in three of the tumors. The size and multiplicity of the nucleoli in these tumor cells suggests that RNA polymerase I could account for the activity which is inhibited by cycloheximide.