Most eukaryotic genes are controlled by a complex array of cis-acting regulatory elements that modulate transcriptional activity. Two major regulatory elements reside in the chicken ovalbumin gene, a steroid-dependent regulatory element (SDRE, -892 to -780) and a negative regulatory element (NRE, -308 to -88). The SDRE is required for responsiveness to estrogen and glucocorticoid. The NRE appears to have the dual role of repressing transcription in the absence of steroids and of cooperating with the SDRE to activate transcription in the presence of steroids. The experiments described herein were designed to investigate the role of the NRE in repressing gene expression. Transfection of OvCAT fusion genes containing deletions in the NRE into primary oviduct cell cultures identified three elements (-308 to -256, -239 to -220, and -174 to -88) that repress transcription. Oligomers corresponding to portions of these elements also independently repress the viral thymidine kinase promoter. Interestingly, the element from -239 to -220 functions mechanistically as a silencer and shares sequence identity with silencers in other genes (TCTCTCCNA). Mobility shift studies indicated that all of the negative elements bind specific protein complexes from oviduct, none of which is appreciably affected by treatment with steroid hormones. However, oviduct-specific proteins bind to the regions from -280 to -252 and from -134 to -88, providing the first identification of potential tissue-specific elements in the ovalbumin gene. These results demonstrate that the region of DNA originally called the NRE is a multifunctional regulatory element that may be involved in several diverse regulatory activities.