The RNA polymerase II large subunit contains tandem copies of the sequence Pro Thr Ser Pro Ser Tyr Ser at its carboxyl terminus, the number of which varies from 26 in yeast to 52 in mice. Our results indicate that the heptapeptide repeat sequence is unique and essential to RNA polymerase II. We have determined that a portion of the heptapeptide repeat domain is essential for viability by constructing and analyzing unidirectional deletions of the carboxy-terminal coding sequence in yeast. Cells containing an RNA polymerase II large subunit with less than 10 complete heptapeptide repeats are inviable, those containing 10-12 complete repeats are conditionally viable, and those with 13 or more complete repeats are unconditionally viable. The inviable deletion mutants studied here have truncated RNA polymerase subunits that are stable, but functionally deficient. Finally, the number of repeat units is polymorphic in wild-type yeast strains. These results have implications for the function of this unusual sequence in transcription.