Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein.