It has been well documented that the repeated CATT(A/T) sequence, localized between -64 and -35 in the human GM-CSF promoter, is required for the promoter activity, and this region likely serves as a core recognition sequence for a cellular transcription factor. However, the transcription factor that interacts with this site was not identified. Here, we report that this element contains a binding site for the nuclear factor YY1, which has not been reported to play a role in the regulation of cytokine gene transcription. Results from transient transfection assays of the Jurkat T cell line revealed that this repeated CATT(A/T) element exhibited enhancer activity when linked to both the human IFN-gamma promoter and the TK promoter. Mutation of the YY1 binding site eliminated about 60% of the enhancer activity of the element. We have found that the YY1 binding site could form two specific DNA-protein complexes, A and B, with Jurkat nuclear proteins in the electrophoretic mobility shift assay and that the binding of these complexes correlates with the enhancer activity. UV cross-linking analysis revealed that the A complex is a multi-protein complex and in addition to YY1, other proteins are required for formation of the protein complex. Cotransfection assays with a YY1 expression vector revealed that overexpression of YY1 resulted in an inhibitory effect on the repeated CATT(A/T) element, indicating that in addition to YY1, cofactors also are required for the activator function of the A complex.