Hepatocytes are the major source of sex hormone-binding globulin (SHBG), a glycoprotein that transports sex steroids in the blood and regulates their access to target tissues. The human SHBG proximal promoter was analyzed by DNase I footprinting, and the functional significance of 6 footprinted regions (FP1-FP6) within the proximal promoter was studied in human HepG2 hepatoblastoma cells. Two footprinted regions (FP1 and FP3) contain binding sites for the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and hepatocyte nuclear factor-4 (HNF-4). In experiments where SHBG promoter-luciferase reporter gene constructs were co-transfected into HepG2 cells with COUP-TF and/or HNF-4 expression vectors, HNF-4 markedly increased transcription, whereas COUP-TF suppressed this probably by displacing HNF-4 from their common FP1-binding site. This COUP-TF/HNF-4-binding site within FP1 includes a TTTAA sequence, located at nucleotides -30/-26 upstream of the transcription start site, which fails to interact with human TFIID, TATA-binding protein in vitro. When this sequence was replaced with an idealized HNF-4-binding site, the transcriptional activity of the promoter increased in HepG2 cells. Taken together, these data imply that an interplay between COUP-TF and HNF-4 at a site within FP1 regulates human SHBG expression and that HNF-4 controls transcription from this TATA-less promoter by somehow substituting for TATA-binding protein in the recruitment of a transcription preinitiation complex.