DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.