The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.