Abstract: The GA-repeat binding protein (GABP) is a ubiquitous transcription factor involved in transcriptional regulation of genes encoding proteins involved in a variety of cellular processes including adipocyte differentiation, mitochondrial respiration, and neuromuscular signaling. GABP is composed of two subunits; the GABPα subunit is a member of the Ets-family of transcription factors, and the unrelated ankyrin repeat containing GABPβ subunit. We previously identified a bidirectional promoter directing the expression of Gabpa (GAA) gene in one direction and ATP Synthase Coupling Factor 6 (Atp5j) (CF6) gene in the other [Chinenov, Y., Coombs, C. and Martin, M. E., 2000a. “Isolation of a bi-directional promoter directing the expression of the mouse GABPα and ATP Synthase Coupling Factor 6 genes. Gene 261:311–320.]. In this study we characterize sequence elements and regulatory factors contributing to the promoter activities of the GAA/CF6 bidirectional promoter. The core of the GAA/CF6 bidirectional promoter is retained within a 400 bp sequence and contains four GABP binding sites, a Sp1/3 binding site and an YY1 binding site. Site-directed mutagenesis demonstrated that while no single factor binding site was essential for promoter activity in either direction, the GA1 site located proximal to the previously mapped transcription start sites functioned cooperatively with the other GABP binding sites and with the Sp1/3 and YY1 sites to provide transcriptional activation of the GAA and CF6 promoters. The other GABP sites and the Sp1/3 and YY1 binding sites were functionally redundant for basal promoter activities in both directions. Electrophoretic mobility shift assays identified multiple DNA-protein complexes containing GABPα, GABPβ, Sp1, Sp3 or YY1 proteins, including one ternary complex containing GABPα, GABPβ and Sp1 proteins. Binding of GABP to the GAA/CF6 bi-directional promoter provides the potential for autoregulation of GABPα expression and confirms the importance of GABP in the coordinate expression of respiratory chain components. [Copyright &y& Elsevier]