The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes three accessory factor-binding sites (AF1, AF2, and AF3), two glucocorticoid receptor-binding sites (GR1 and GR2), and a cAMP response element. All of these elements, and the proteins that bind to them, are required for a complete glucocorticoid response. The PEPCK promoter also contains a retinoic acid response unit (RARU) that consists of two retinoic acid response elements (RARE1 and RARE2) that bind retinoic acid receptor/9-cis-retinoic acid receptor heterodimers. The sequences of RARE1 and RARE2 coincide with those for AF1 and AF3, respectively. Thus, the PEPCK promoter can mediate different hormone responses through hormone response units that utilize common elements, but that bind different sets of proteins. We reasoned that each response might require a unique structural assembly and therefore tested how various arrangements of the PEPCK promoter affect the actions of either glucocorticoids or retinoic acid. The activation of the PEPCK gene in response to glucocorticoids requires a specific set of cis-acting elements that must be precisely positioned within the GRU. The distance between AF2 and GR1 is critical for the glucocorticoid response, and since the AF2 factor, HNF3, interacts with GR in vitro, this protein-protein interaction may be important for the glucocorticoid response. By contrast, the distance and orientation requirements of AF1 and AF3 with respect to GR1 are more flexible. In the case of the RARU, although the relative positions of RARE1 and RARE2 are important for the retinoic acid response, more tolerance for distance and stereospecific alignment between RARE1 and RARE2 is allowed. In addition, we show that the GRU and the RARU can act as a module, within a restricted region, in the context of the PEPCK promoter and in limited contexts of a heterologous promoter. These observations suggest that the structural requirements of the GRU and the RARU are different, and this may have important implications for how multiple hormonal signals are integrated through this promoter.