Glucocorticoids serve as important therapeutic agents in diseases of inflammation, but clinical use, especially in advanced septic shock, remains controversial because of the unpredictable response. Prior studies correlate human glucocorticoid receptor (hGR) isoforms with a decreased response to steroid therapy. Further analysis of additional hGR isoforms may improve the understanding of the steroid response. Ninety-seven human volunteers' blood samples were surveyed for hGR isoforms. An isoform matching National Center for Biotechnology Informatics (NCBI) hGRα (hGR NCBI) served as a reference. Two isoforms were of particular interest-one isoform had three nonsynonymous single-nucleotide polymorphisms (SNPs) (hGR NS-1), and the second had a single-nucleotide deletion (hGR DL-1) resulting in a truncated protein. Transactivation potentials were measured using a luciferase reporter assay. Human glucocorticoid receptor NS-1 had activity more than twice of hGR NCBI, whereas hGR DL-1 demonstrated less than 10% of the activity of hGR NCBI. Cotransfection of two isoforms revealed that the presence of hGR NS-1 increased transactivation potential, whereas hGR DL-1 decreased activity. Synthetic constructs isolating individual and paired SNPs of hGR NS-1 were created to identify the SNP responsible for hyperactivity. Transactivation studies revealed a SNP within the ligand-binding domain exerted the greatest influence over hyperactivity. In evaluating the response to hydrocortisone, hGR NCBI and hGR NS-1 displayed an increased dose-dependent response, but hGR NS-1 had a response more than twice hGR NCBI. Characterization of the novel hyperactive hGR NS-1 provides insight into a possible mechanism underlying the unpredictable response to steroid treatment.