Hormone-responsive genes rely on complex regulatory elements known as hormone response units to integrate various regulatory signals. Characterization of the steroid-dependent regulatory element (SDRE) in the check ovalbumin gene (-892 to -796) suggests that it functions as a hormone response unit. Previous studies using gel mobility shift assays and several types of footprinting analyses demonstrated that proteins bind to this entire element in vitro even in the absence of steroid hormones. However, the genomic footprinting experiments described herein indicate that the binding of three different proteins or protein complexes to the SDRE requires estrogen and corticosterone, suggesting that the chromatin structure of this site is restricted in vivo. Transfection experiments using linker scanning and point mutations support the contention that the binding of these three complexes is essential for induction of the ovalbumin gene by steroid hormones. In addition, functional analyses suggest that a fourth complex is also necessary for maximal induction. These and other data suggest that the SDRE functions as a hormone response unit to coordinate signals generated by two steroid hormones.