The peptide hormone oxytocin is highly expressed in the hypothalamus within only a small number of magnocellular neurons. However, it is also expressed in a much larger number of cells in the bovine corpus luteum at high levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studies, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been identified as the bovine homologue of the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The second protein is here characterized as the bovine homologue of a tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The relative expression of these two factors during luteal development correlates with the level of luteal oxytocin gene expression, with SF-1 being the factor binding to the promoter of the oxytocin gene when this promoter is activated. Cotransfection experiments using the murine testicular cell line TM4 show that SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly by transducing a stimulatory signal to the RNA polymerase.