Mammalian RNA polymerase I (Pol I) is a multisubunit enzyme that is decorated with accessory proteins, termed PAFs (polymerase-associated factors). The presence or absence of distinct PAFs may account for the functional differences of distinct fractions of cellular Pol I, and suggests that PAFs could be targets of regulatory pathways. Here we describe and functionally characterize PAF67, a novel 67 kDa protein that is tightly associated with a subpopulation of cellular Pol I. Both PAF67-containing and -deficient Pol I transcribe non-specific templates with similar efficiency, however, only the enzyme that contains PAF67 is capable of specifically transcribing rDNA templates. PAF67 co-localizes with Pol I in the nucleolus at sites of active rDNA transcription, indicating that PAF67 serves a role in rDNA transcription initiation. The results suggest that association of PAF67 with the 'core' enzyme endows Pol I with the capability to assemble into a productive transcription initiation complex at the rDNA promoter.