The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two factors play in transcriptional regulation of this gene. To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter. The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts. We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication. CPF1 acts as a negative regulator, modulating the overall induction response. Alleviation of repression through CPF1 requires passage through the S phase. Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.