When protein biosynthesis is inhibited by either cycloheximide of puromycine, the nucleolar RNA synthesis of Ehrlich ascites tumor cells decreases by approximately 70% within 1 h, while the removal of these protein synthesis inhibitors causes a rapid recovery of nucleolar RNA synthesis, largely within 1 h. A similar pattern of decrease and recovery of endogenous RNA polymerase activity in isolated nucleoli or in nuclei (in the presence of alpha-amanitin) may be demonstrated after addition and removal of these drugs. Analysis of the molecular species of RNA polymerase I on a phosphocellulose column indicates that only the IB form of the enzyme decreases in the nucleoli of drug-treated cells and recovers quickly after resumption of protein synthesis. The finding that the activity of the IB form enzyme remains unchanged in the whole nuclei indicates that during cessation of protein synthesis RNA polymerase IB is either released from the nucleoli into the extranucleolar compartment or becomes so loosely bound to the nucleoli that it is leached out from the nucleoli during their isolation. By using a system of assaying free, nucleolar-template bound and total RNA polymerase I activities, data supporting the above interpretation have been obtained. Namely, in isolated nuclei free enzyme activity increases with a concomitant decrease in bound enzyme activity during protein synthesis inhibition, while the total enzyme activity remains unchanged. In isolated nucleoli, both total and bound enzyme activities decreases on protein synthesis inhibition but recover quickly on its resumption. The putative bound enzyme, fractionated with the aid of actinomycin D, is exclusively IB form, whereas the unbound enzyme consists of both IA and IB forms as previously demonstrated (1). No conversion of IB form polymerase to IA form was noted on prolonged sonication in our system. The levels of ATP and GTP in the cell did not change appreciably either during cessation or resumption of protein synthesis in these cells. The data support the previous conclusion that some short-lived protein(s) is required to maintain the normal level of ribosomal RNA transcription (2) and further suggest that the protein is required to facilitate reinitiation of the transcription by RNA polymerase IB in the nucleolus.