DNA regulatory elements frequently harbor multiple recognition sites for several transcriptional activators. The response mounted from such compound response elements is often more pronounced than the simple sum of effects observed at single binding sites. The determinants of such transcriptional synergy and its control, however, are poorly understood. Through a genetic approach, we have uncovered a novel protein motif that limits the transcriptional synergy of multiple DNA-binding regulators. Disruption of these conserved synergy control motifs (SC motifs) selectively increases activity at compound, but not single, response elements. Although isolated SC motifs do not regulate transcription when tethered to DNA, their transfer to an activator lacking them is sufficient to impose limits on synergy. Mechanistic analysis of the two SC motifs found in the glucocorticoid receptor N-terminal region reveals that they function irrespective of the arrangement of the receptor binding sites or their distance from the transcription start site. Proper function, however, requires the receptor's ligand-binding domain and an engaged dimer interface. Notably, the motifs are not functional in yeast and do not alter the effect of p160 coactivators, suggesting that they require other nonconserved components to operate. Many activators across multiple classes harbor seemingly unrelated negative regulatory regions. The presence of SC motifs within them, however, suggests a common function and identifies SC motifs as critical elements of a general mechanism to modulate higher-order interactions among transcriptional regulators.