The human adenine nucleotide translocator‐2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides −79 and −68 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1‐mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1‐mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox‐mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox‐bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species.