The mineralocorticoid receptor (MR) and its closest relative the glucocorticoid receptor (GR) both act as ligand-bound transcription factors at a joint hormone response element. However, while activated MR regulates electrolyte and water homeostasis and is involved in pathophysiological remodeling processes in the renocardiovascular system, GR elicits very different effects. Consequently, the existence of additional DNA-binding elements has been postulated. Previously, we showed that ligand-bound MR mediates some pathophysiological effects by specifically up-regulating the epidermal growth factor receptor (EGFR) via enhanced EGFR promoter activity. To decipher the mechanism for the MR-EGFR-promoter interaction, we performed reporter-gene-assays with deletion constructs of the EGFR promoter and identified a 65 bp mineralocorticoid receptor binding element (MRE) which does not interact with GR. Because an in vitro transcription factor binding assay with this MRE indicated that activated MR does not specifically bind to MRE by itself we looked for additional binding sites on our MRE. Using bioinformatical tools we identified an SP1 binding motif within the MRE sequence. Specific and direct binding of SP1 to MRE could be confirmed by EMSA with a biotinylated MRE probe and recombinant hSP1. Furthermore, in the presence of SP1 MR showed specific binding to MRE in our in vitro transcription factor binding assay. Accordingly, inhibition of SP1 abolished MR-induced induction of EGFR promoter activity in a MRE reporter gene assay. A more detailed analysis of the MR-SP1-MRE interaction and a screening of the genome for further MRE regulated genes seem worthwhile for developing strategies to prevent pathophysiological aldosterone effects.