Objective: The mineralocorticoid (MR) and glucocorticoid receptor (GR) belong to the steroid receptor family. Both act as ligand inducible transcription factors which bind in the active form at the glucocorticoid hormone response element (GRE) in the promoter region of genes. Although MR and GR bind to the same GRE, these receptors mediate different effects in vivo. The MR regulates water and salt homeostasis whereas GR mediates metabolic homeostasis and anti-inflammatory actions. The aim of the present study was to compare directly binding behaviour of MR and GR to GRE. Methods: Binding of activated MR/GR to GRE was analysed using an ELISA based transcription factor GRE binding assay. This method was performed with nuclear cell extracts (NCE) and in vitro synthesized hMR, hGR and LacZ (negative control). All three utilised plasmid vectors contain the same XPRESS-flag. The transcription factors bound to a biotinylated GRE probe could be detected by a specific anti-XPRESS antibody and a horseradish peroxidase-coupled secondary antibody followed by a specific coloured substrate reaction. Results: Specific binding of in vitro synthesized hMR and hGR to GRE show similar affinity and capacity. The addition of saline containing NCE leads to reduced attachment of hMR and hGR to GRE. Yet hMR binding is reduced than the hGR attachment when using high saline containing NCE. In comparison to hGR, hMR binds with reduced affinity and capacity to GRE when using desalted NCE. Conclusion: Although hMR and hGR vary only slightly in the DNA binding domain, a change in micro milieu induces different attachment behaviour at GRE. HMR binds better than hGR under high saline conditions. This could cause a different activation of gene transcription by hMR/hGR under saline conditions and may be one explanation for the different effects in vivo.