Previous experiments using mammalian cells suggested that after each round of transcription, RNA polymerase I (Pol I) dissociates into subunits that leave and reenter the nucleolus as individual subunits, before formation of a new initiation complex. In this study, we show that the size and subunit composition of Pol I did not change significantly when Pol I was not engaged in rRNA transcription, brought about by either the absence of Pol I-specific rDNA template or specific inhibition of the transcription initiation step that requires Rrn3p. In fact, Pol I purified from cells completely lacking rDNA repeats was more active than when purified from wild-type cells in an in vitro transcription system designed to assay active Pol I-Rrn3p complexes. Furthermore, measurements of the exchange of A135 and A190 subunits between preexistent Pol I and newly synthesized Pol I showed that these two largest subunits of Pol I do not disassociate through many rounds of transcription in vivo. Thus, Pol I is not a dynamic protein complex but rather a stable enzyme.