In the past decade, investigation into steroid hormone signaling has focused on the mechanisms of steroid hormone receptors as they act as signaling molecules and transcription factors in cells. However, the majority of hormone-responsive genes are not directly regulated by hormone receptors. These genes are termed secondary response genes. To explore the molecular mechanisms by which the steroid hormone estrogen regulates secondary response genes, the ovalbumin (Ov) gene was analyzed. Three protein/protein complexes (Chirp-I, -II, -III), which do not contain the estrogen receptor, are induced by estrogen to bind to the 5' flanking region of the Ov gene. The Chirp-III DNA-binding site, which is required for estrogen induction, binds a complex of proteins that contains the estrogen-inducible transcription factor EF1. Experiments undertaken to identify proteins complexed with EF1 led to the elucidation of a novel mechanism of action of upstream stimulatory factor-1 (USF-1), which involves its tethering to the Ov gene 5' flanking region by EF1. Gel mobility shift assays and co-immunoprecipitation experiments identify USF-1 as a component of Chirp-III. However, USF-1 is not able to bind to the Chirp-III site independently. In addition, USF-1 overexpression is able to induce Ov gene promoter activity in transfection experiments. USF-1 can also potentiate the induction of the Ov gene by the transcription factor EF1. Moreover, mutating the EF1 binding sites in the 5' flanking region of the Ov gene abrogates induction of the gene by USF-1. These data begin to establish a molecular mechanism by which hormone-inducible transcription factors and ubiquitous transcription factors cooperate to regulate estrogen-induced secondary response gene expression.