GATA-6 and GATA-4 are members of a family of transcription factors (GATA 1-6) that share conserved zinc-finger DNA binding domains. Using semiquantitative RT-PCR, we found that the human adrenal expresses mRNA for GATA-6 but not GATA-4. A recent study showed GATA-6 expression in the adrenal reticularis, the source of adrenal androgens. To investigate the role of GATA-6 in regulation of adrenal cell steroidogenesis, luciferase reporter constructs containing the 5'-flanking DNA from steroidogenic acute regulatory protein, cholesterol side-chain cleavage (CYP11A), 17alpha-hydroxylase (CYP17), and dehydroepiandrosterone-sulfotransferase (SULT2A1) were cotransfected with an expression vector containing GATA-6 into adrenal NCI-H295R cells and nonsteroidogenic HEK293 cells. All promoter/reporter constructs were increased by GATA-6 in the adrenal model. However, in the HEK293 cells only SULT2A1 reporter activity was increased by GATA-6. One key difference between H295R and HEK293 cell lines is the differential expression of steroidogenic factor 1 (SF1). Transfection of HEK293 cells with both GATA-6 and SF1 significantly increased transcriptional activation of all reporter constructs above the effect of GATA-6 or SF1 alone. To determine whether the action of GATA-6 required SF1, we transfected HEK293 cells with each promoter construct plus and minus GATA-6, SF1, and/or the orphan nuclear repressor DAX1. DAX1 opposed SF1-activated transcription of many genes and abolished the GATA-6/SF1 ability to increase reporter activity. These results suggest that the adrenal uses GATA-6 to enhance transcription of steroid-metabolizing enzymes needed to produce dehydroepiandrosterone sulfate. Additionally, GATA-6 works in synergy with SF1 to maximally increase expression of enzymes needed to produce adrenal androgens.