Monoclonal antibodies that recognize specific carboxyl-terminal domain (CTD) phosphoepitopes were used to examine CTD phosphorylation in yeast cells lacking carboxyl-terminal domain kinase I (CTDK-I). We show that deletion of the kinase subunit CTK1 results in an increase in phosphorylation of serine in position 5 (Ser(5)) of the CTD repeat (Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7)) during logarithmic growth. This result indicates that CTDK-I negatively regulates CTD Ser(5) phosphorylation. We also show that CTK1 deletion (ctk1Delta) eliminates the transient increase in CTD serine 2 (Ser(2)) phosphorylation observed during the diauxic shift. This result suggests that CTDK-I may play a direct role in phosphorylating CTD Ser(2) in response to nutrient depletion. Northern blot analysis was used to show that genes normally induced during the diauxic shift are not properly induced in a ctk1Delta strain. Glycogen synthase (GSY2) and cytosolic catalase (CTT1) mRNA levels increase about 10-fold in wild-type cells, but this increase is not observed in ctk1Delta cells suggesting that increased message levels may require Ser(2) phosphorylation. Heat shock also induces Ser(2) phosphorylation, but we show here that this change in CTD modification and an accompanying induction of heat shock gene expression is independent of CTDK-I. The observation that SSA3/SSA4 expression is increased in ctk1Delta cells grown at normal temperature suggests a possible role for CTDK-I in transcription repression. We discuss several possible positive and negative roles for CTDK-I in regulating CTD phosphorylation and gene expression.