Glucocorticoid resistance is a rare, familial, or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition ranges from completely asymptomatic to severe hyperandrogenism, fatigue, and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the human glucocorticoid receptor-α (hGRα) gene, which impair the ability of the receptor to transduce the glucocorticoid signal. We systematically investigated the molecular mechanisms through which natural, ligand-binding domain hGRα mutants, including hGRαI559N, hGRαV571A, hGRαD641V, hGRαV729I, and hGRαI747M, produce a defective signal and determined whether their differential effects on hGRα function might account for the type of genetic transmission of the disorder and the variable clinical phenotype of the affected subjects. Our findings suggest that all five mutant receptors studied have ligand-binding domains with decreased intrinsic transcriptional activity. Unlike hGRαI559N and I747M previously shown to exert a dominant negative effect upon the transcriptional activity of hGRα, hGRαV571A, D641V, and V729I do not have such an effect. All five mutants studied demonstrate varying degrees of decreased affinity for the ligand in a standard dexamethasone binding assay, but preserve their ability to bind DNA. The nondominant negative mutants, hGRαV571A, D641V, and V729I, show delayed translocation into the nucleus after exposure to ligand. Finally, hGRαI559N, V571A, D641V, and V729I display an abnormal interaction with the glucocorticoid receptor-interacting protein-1 coactivator in vitro, as this was previously shown also for hGRαI747M. We conclude that each of the above hGRα mutations imparts different functional defects upon the glucocorticoid signal transduction pathway, which explains the autosomal recessive or dominant transmission of the disorder, but might only explain in part its variable clinical phenotype.