The ovalbumin (Ov) gene contains a number of regulatory elements that control its transcriptional activity and restrict expression to avian oviduct. One major regulatory region, the steroid-dependent regulatory element (SDRE), is required for induction by estrogen and corticosterone. Another region, the negative regulatory element (NRE), downstream of the SDRE, acts primarily to repress gene expression. In addition, experiments within indicate that the binding site for the COUP transcription factor (COUP-TF) is also required for Ov gene transcription. To examine the interactions involving the SDRE, the NRE, and the COUP binding sites on Ov gene transcription, mutations in these regions were made and transfected into primary oviduct cell cultures. These experiments show that without the NRE, the SDRE is sufficient for induction by estrogen and corticosterone, irrespective of the COUP site. However, with the NRE intact, the COUP site is required for steroid induction, although without the NRE, the COUP site attenuates transcriptional activity. More interestingly, overexpression of COUP-TF1 with the Ov wild-type reporter construct alleviates the requirement for steroid hormones. These results demonstrate that the COUP site is essential and has a dual role in Ov gene transcription and that steroid hormones might directly or indirectly regulate the activity of COUP-TF1.