The expression of some nuclear genes in Saccharomyces cerevisiae, such as the CIT2 gene, which encodes a glyoxylate cycle isoform of citrate synthase, is responsive to the functional state of mitochondria. Previous studies identified a basic helix-loop-helix-leucine zipper (bHLH/Zip) transcription factor encoded by the RTG1 gene that is required for both basal expression of the CIT2 gene and its increased expression in respiratory-deficient cells. Here, we describe the cloning and characterization of RTG3, a gene encoding a 54-kDa bHLH/Zip protein that is also required for CIT2 expression. Rtg3p binds together with Rtg1p to two identical sites oriented as inverted repeats 28 bp apart in a regulatory upstream activation sequence element (UASr) in the CIT2 promoter. The core binding site for the Rtg1p-Rtg3p heterodimer is 5'-GGTCAC-3', which differs from the canonical E-box site, CANNTG, to which most other bHLH proteins bind. We demonstrate that both of the Rtg1p-Rtg3p binding sites in the UAS(r) element are required in vivo and act synergistically for CIT2 expression. The basic region of Rtg3p conforms well to the basic region of most bHLH proteins, whereas the basic region of Rtg1p does not. These findings suggest that the Rtg1p-Rtg3p complex interacts in a novel way with its DNA target sites.