We have addressed whether the intrinsic 3'-->5' nuclease activity of human RNA polymerase II (pol II) can proofread during transcription in vitro. In the presence of SII, a protein that stimulates the nuclease activity, pol II quantitatively removed misincorporated nucleotides from the nascent transcript during rapid chain extension. The basis of discrimination between the correct and incorrect base was the slow addition of the next nucleotide to the mismatched terminus. Incorporation of inosine monophosphate inhibited next nucleotide addition by a similar magnitude as a mismatched base. We used this finding to demonstrate that addition of SII to a transcription reaction dramatically altered the RNA base content, reflecting the stable incorporation of more "correct" (GMP) and fewer "incorrect" (IMP) nucleotides.