As is the case with many eukaryotic genes, regulation of the chick ovalbumin (Ov) gene involves both positive and negative modulation. Recent studies indicate that positive regulation by steroids entails binding of several proteins to a hormone-response unit called the steroid-dependent regulatory element (SDRE; -892 to -780). In addition, gene activity is suppressed by factor(s) acting through the negative regulatory element (NRE; -308 to -88). Previous data suggested that the NRE is composed of multiple, independently acting negative elements. The goal of the present studies was to define more precisely the locations of these negative elements and to investigate their functional interactions. Transfection analyses of linker scanning mutants revealed a strong repressor site, designated the COUP- adjacent repressor (CAR) site, located between -119 and -111. Gel mobility shift analyses with the CAR element suggested that it may play a role in the developmental regulation of the Ov gene. A weaker repressor element was also identified at about -275. Surprisingly, two positive sites were found, one of which is the binding site for the estrogen-responsive transcription factor δ-EF1. These results demonstrate that the Ov NRE contains not only sites responsible for the repression of the gene but also a positive element that is required for responsiveness to steroid hormones.