The QCR8 gene of the yeast K1uyveromyces lactis is transcriptionally regulated by the carbon source in the growth medium. Deletion analysis of the KlQCR8 promoter shows that an element located between -144 bp and -113 bp specifically controls induction of QCR8 gene expression on non-fermentable carbon sources. Specific and differential protein-binding to the activating sequence was observed with extracts from glucose- and ethanol/glycerol-grown cells. Induction of the reporter gene and protein-binding was dependent on the presence of a functional KlCAT8 gene, suggesting that, in K. lactis, K1Cat8p acts in the transcriptional regulation of respiratory function. The activating element contains no other known regulatory sites but two elements required for RNA holoenzyme functioning, raising the intriguing possibility of carbon source-dependent regulation by a subunit of the RNA polymerase holoenzyme in K. lactis.