To study the regulation of the human glucocorticoid receptor (hGR), we characterized the promoter region by primer extension, S1 nuclease mapping and by DNA sequencing. We found that the promoter is extremely G + C rich (72% GC content) and contains a "TAATA" and a "CAT" box, eight "GGGCGG", three "CCGCCC" and two "CACCC" motifs and a motif similar to the glucocorticoid responsive element (GRE) which included two interchanged nucleotides "TCTTGT". In contrast to other steroid receptor genes, exon I or GHGR contains the major part of the 5' non-coding sequences of hGR mRNA while exon II contains coding sequences for the first 394 amino acid residues of the A/B region of hGR. The major transcriptional start site was found to be 134 bp upstream of the ATG initiation codon. Transfection of HeLa cells with plasmids containing various deletions of GHGR promoter fused to a promoterless CAT vector suggested the region between -470 and -1030, at the 5' end of the mRNA start site, to contain sequences required for down regulation by hormone.