A 2954 bp region of the guinea pig estrogen sulfotransferase gene was cloned and sequenced including 2888 nt upstream of the cap site. This represents the first cloning of the promoter region of a steroid sulfotransferase gene. The 5'-flanking region was found to contain a conventional TATA box variant and sequences homologous to estrogen and glucocorticoid response elements. Gel mobility shift assays detected the presence of nuclear proteins in adrenocortical SW-13 and Y-1 cells that bind specifically to 30 mer DNA sequences containing either estrogen or glucocorticoid response elements. In contrast, gel shift experiments using 3T3 fibroblast cells failed to demonstrate similarly upshifted bands. Block deletion studies indicated that regulation of basal estrogen sulfotransferase promoter activity was located within the first 1000 bp upstream of the transcription initiation site.