The GATA transcription factors are crucial regulators of cell-specific gene expression in many tissues. GATA proteins are abundantly expressed in gonads of several species. In vertebrates, GATA factors are expressed from the onset of gonadal development and are later found in multiple cell lineages of both the testis and ovary. GATA factors activate transcription of several gonadal genes including the hormone-encoding genes Müllerian inhibiting substance (MIS) and inhibin alpha and genes involved in steroidogenesis like P450 aromatase (Cyp 19) and steroidogenic acute regulatory protein. GATA factors also contribute to cell-specific gonadal gene expression through cooperative interactions with other transcription factors such as the orphan nuclear receptor steroidogenic factor-1. GATA transcriptional activity is also modulated by two multitype zinc finger proteins called the Friend of GATA (FOG) proteins, which were cloned as GATA-specific cofactors. The FOG proteins (FOG-1 and FOG-2) can act as either enhancers or repressors of GATA transcriptional activity, depending on the cell and promoter context. We now report that the FOG proteins are coexpressed with GATA factors in testicular cells in which they differentially repress the promoter activities of several GATA-dependent target genes. These findings implicate the FOG proteins in the regulation of GATA-dependent gene transcription in the gonads.