When mammalian cells are exposed to cisplatin or ultraviolet irradiation, the RNA polymerase II (RNAP II) large subunit becomes ubiquitinated and is subsequently degraded via the proteasomal pathway. Using a DNA template immobilized on magnetic beads in an in vitro transcription reaction, we showed that a pause of the elongating RNAP II complex caused by nucleotide starvation induced the ubiquitination of the stalled RNAP II. The ubiquitinated RNAP II dissociated from the ternary complex when transcription was allowed to resume. The dissociated (free) RNAP II remained ubiquitinated. The proteasome inhibitor MG132 increased the accumulation of ubiquitinated free RNAP II but did not affect the amount of ubiquitinated, template-bound RNAP II, indicating that the ubiquitinated RNAP II was displaced from the template and then degraded by the proteasomes. Our work shows that the elongation complex that was stalled at the template by nucleotide starvation is targeted by the ubiquitin-conjugating system and that ubiquitination facilitates displacement of the stalled RNAP II from the template. Our findings together with the findings by others that DNA damaging agents induced the ubiquitination in mammalian cells that are nucleotide excision repair competent, suggest that the RNAP II ubiquitination may have a role in the regulation of transcription-coupled DNA repair.