A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.