Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32β) that led to the demonstration of two human Pol III isoforms (Pol IIIα and Pol IIIβ). RPC32β-containing Pol IIIβ is ubiquitously expressed and essential for growth of human cells. RPC32α-containing Pol IIIα is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32α expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32α in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.T ranscription in eukaryotes is mediated by three nuclear DNAdependent RNA polymerases (Pol I, Pol II, and Pol III) (1, 2). Pol III directs transcription of small noncoding RNAs that are involved in translation, splicing, and other cellular processes. Transcription by Pol III is directed by at least three distinct promoter types. Type 1 (5S RNA) and type 2 [tRNA, Alu RNA, and adenoviral viral-associated (VA) RNA] promoters are internal to the gene. Type 3 (U6 and 7SK RNA) promoters are located 5′ to the transcription initiation site (3). The transcription factors that directly recognize these promoters [type 1 by gene-specific TFIIIA and general initiation factor TFIIIC; type 2 by TFIIIC; and type 3 by gene-specific PSE-binding transcription factor/small nuclear RNA-activating protein complex (PTF/SNAPc)] have been well characterized and shown to recruit general initiation factor TFIIIB to their cognate promoters (reviewed in ref. 4). Overall, the multisubunit compositions of TFIIIC and TFIIIB have been conserved from yeast to human (5, 6), but two distinct isoforms of TFIIIB have been identified in human cells-one (TFIIIB-β) active in transcription of type 1 and type 2 promoters and one (TFIIIB-α) active in transcription of type 3 promoters (7). This functional difference reflects the presence of BRF1 in TFIIIB-β and of its paralogue BRF2 in TFIIIB-α (8, 9).Pol III is highly conserved from yeast to humans and composed of 17 subunits. Of these subunits, five are common to all three polymerases, two are shared by Pol I and Pol III, and five are paralogous to subunits found in Pol I and Pol II. However, five subunits are specific to Pol III without a counterpart in Pol I or Pol II (reviewed in refs. 5 and 10). Three of these five Pol III-specific subunits (RPC32, RPC39, and RPC62) form a dissociable ternary subcomplex that is specifically required for transcription initiation (11). This ternary complex...