We recently reported that the 19S regulatory subunit of the yeast 26S proteasome stimulates transcription elongation by RNA polymerase II. However, because of basic differences between yeast and mammals in the components and cellular location of the proteasome, it is crucial to assess whether this is a general phenomenon. Here we address this question and demonstrate that (1) the nonproteolytic activity of the 19S (PA700) complex of the proteasome is required for efficient activated transcription in the mammalian in vitro system, (2) this requirement applies to both natural and artificial activators, and (3) highly purified PA700 can provide this activity. In vitro transcription assays using HeLa cell nuclear extracts reveal that antibodies against human Trip1p/Rpt6 (mammalian Sug1p), one of the six ATPases in the PA700, significantly inhibit activated transcription. Similarly, immunodepletion of the PA700 from the extract also significantly reduces activated, but not basal, transcription and add-back of the highly purified mammalian PA700 restores the activity. Finally, inhibitors of the proteasome's peptidase activities do not affect transcription although the peptidase activity is almost completely inhibited. These findings indicate that the requirement for a nonproteolytic activity of the 19S complex in transcription is general in eukaryotes.