NC2 is a heterodimeric regulator of transcription that plays both positive and negative roles in vivo. Here we show that the α and β subunits of yeast NC2 are not always associated in a tight complex. Rather, their association is regulated, in particular by glucose depletion. Indeed, stable NC2 α/β complexes can only be purified from cells after the diauxic shift when glucose has been depleted from the growth medium. In vivo, the presence of NC2 α, but not NC2 β, at promoters generally correlates with the presence of TBP and transcriptional activity. In contrast, increased presence of NC2 β relative to TBP correlates with transcriptional repression. NC2 is regulated by phosphorylation. We found that mutation of genes encoding casein kinase II (CKII) subunits as well as potential CKII phosphorylation sites in NC2 α and β affected gene repression. Interestingly, NC2-dependent repression in the phosphorylation site mutants was only perturbed in high glucose when NC2 β and NC2 α are not associated, but not after the diauxic shift when NC2 α and β form stable complexes. Thus, the separation of NC2 α and β function indicated by these mutants also supports the existence of multiple NC2 complexes with different functions in transcription.