Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.