Studies on the human glucocorticoid receptor (GR) promoter were carried out so as to understand the regulation of GR expression. A -2738 to +19 fragment of the human GR promoter was used to identify important regulatory elements involved in the control in GR expression in NIH 3T3 cells (mouse fibroblasts) and HeLa cells (human cervical carcinoma cells). Important regulatory domains in the distal region of the human GR promoter were identified by sequential 5' end deletion analysis. A region between -2490 and -2025 contributed 50% of the measured transcriptional activity to the promoter. Using DNase I footprint analysis, four sites in this region were identified: -2362 to -2339 (mouse footprint, mFP); -2301 to -2293 (distal YY1, dYY1); -2130 to -2122 (middle YY1, mYY1); and, -2086 to -2078 (proximal YY1, pYY1). Three sites contained an identical core sequence, CCAAGATGG and were identified as Yin Yang 1 (YY1) binding sites. The site located at -2362 to -2339 was footprinted in NIH 3T3 cells only. The sequence of this site is a direct repeat with a 2-nucleotide spacer region, and it does not share homology with any known transcription factor binding sites. Computer analysis of the entire promoter sequence revealed an additional YY1 site located at -260 to -249 (initiator YY1, iYY1) with the sequence CTCCTCCATTTTG. Electrophoretic mobility supershift assays, with an anti-YY1 antibody, were used to confirm YY1 binding to these four putative YY1 binding sites. Site-directed deletion of all three upstream YY1 sites but not the iYY1 site, or the iYY1 site alone, showed a approximately 60% decrease in transcriptional activity of the hGR promoter in HeLa cells but had no effect in NIH 3T3 cells. A similar (50%) decrease in the expression of a full-length hGR/luciferase reporter gene was obtained when HeLa cells were cotransfected with a full-length antisense YY1 expression plasmid. Additionally, a region between -1841 and -1689 contributed to hGR promoter activity in both cell types tested. An Sp1 binding site was identified in this region (-1748 to -1733) by DNase I footprint and mobility supershift analyses. The presence of four YY1 binding sites in the human GR promoter suggests that these sites play a critical role in GR gene regulation.