The fidelity of yeast RNA polymerase II (Pol II) was assessed in vivo with an assay in which errors in transcription of can1-100, a nonsense allele of CAN1, result in enhanced sensitivity to the toxic arginine analog canavanine. The Pol II accessory factor TFIIS has been proposed to play a role in transcript editing by stimulating the intrinsic nuclease activity of the RNA polymerase. However, deletion of DST1, the gene encoding the yeast homolog of TFIIS, had only a small effect on transcriptional fidelity, as determined by this assay. In contrast, strains containing a deletion of RPB9, which encodes a small core subunit of Pol II, were found to engage in error-prone transcription. rpb9Delta strains also had increased steady-state levels of can1-100 mRNA, consistent with transcriptional errors that decrease the normal sensitivity of the can1-100 transcript to nonsense-mediated decay, a pathway that degrades mRNAs with premature stop codons. Sequences of cDNAs from rpb9Delta strains confirmed a significantly increased occurrence of transcriptional substitutions and insertions. These results suggest that Rpb9 plays an important role in maintaining transcriptional fidelity, whereas TFIIS may serve a different primary purpose.