RNA polymerase (pol) III contains a dissociable subcomplex that is required for initiation, but not for elongation or termination of transcription. This subcomplex is composed of subunits RPC3, RPC6 and RPC7, and interacts with TFIIIB, a factor that is necessary and sufficient to support accurate pol III transcription in vitro . Direct binding of TFIIIB to RPC6 is believed to recruit pol III to its genetic templates. However, this has never been tested in vivo . Here we combine chromatin immunoprecipitation with RNA interference to demonstrate that the RPC3/6/7 subcomplex is required for pol III recruitment in mammalian cells. Specific knockdown of RPC6 by RNAi results in post-transcriptional depletion of the other components of the subcomplex, RPC3 and RPC7, without destabilizing core pol III subunits or TFIIIB. The resultant core enzyme is defective in associating with TFIIIB and target genes in vivo . Promoter occupancy by pol II is unaffected, despite sharing five subunits with the pol III core. These observations provide evidence for the validity in vivo of the model for pol III recruitment that was built on biochemical data.