CONTEXT Primary generalized glucocorticoid resistance is a rare genetic condition characterized by partial end-organ insensitivity to glucocorticoids. Most affected subjects present with clinical manifestations of mineralocorticoid and androgen excess. The condition has been associated with inactivating mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGRα action, thereby reducing tissue sensitivity to glucocorticoids. OBJECTIVE ΤHE aim of our study was to investigate the molecular mechanisms through which one previously described natural heterozygous V423A mutation, the second mutation detected in the DNA-binding domain (DBD) of the hGRα, affects glucocorticoid signal transduction. DESIGN AND RESULTS Compared with the wild-type receptor, hGRαV423A demonstrated a 72% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone. The hGRαV423A receptor showed a significant reduction in its ability to bind to glucocorticoid-response elements of glucocorticoid-responsive genes, owing to structural alterations of the DBD confirmed by computer-based structural analysis. In addition, hGRαV423A demonstrated a 2.6-fold delay in nuclear translocation following exposure to the ligand, although it did not exert a dominant negative effect on the wild-type hGRα, had a similar affinity to the ligand with the wild-type receptor, and displayed a normal interaction with the GRIP1 coactivator in vitro. CONCLUSIONS The natural mutant receptor hGRαV423A causes primary generalized glucocorticoid resistance by affecting multiple steps in the cascade of glucocorticoid receptor action, which primarily involve decreased ability to bind to target glucocorticoid response elements and delayed translocation into the nucleus.