Glucocorticoids remain a recommended therapy in advanced septic shock despite the often unpredictable response, and our understanding of the mechanisms regulating the steroid and stress response remains limited. Since the initial sequencing of the human glucocorticoid receptor α and β gene (hGRα and hGRβ), only three additional splice variants have been identified--all of which have been postulated to contribute to steroid resistance. During a survey of 97 healthy humans' blood, we identified two novel hGR splice isoforms (hGR-S1 and hGR-S1(-349A) retaining intron H between exons 8 and 9. Human GR-S1(-349A) contained a base deletion causing an early termination and a truncated protein of 118 amino acids, whereas hGR-S1 had an early termination occurring within intron H and resulted in a 745-amino acid protein. Both isoforms had decreased transactivation potentials compared with hGRα when tested in the absence of exogenous steroids. However, after treating with exogenous steroids, dose-response studies showed hGR-S1(-349A) had a substantial augmentation in activity at higher concentrations of hydrocortisone and methylprednisolone when compared with hGRα, whereas hGR-S1 did not. Removal of the 3' untranslated region (3'UTR) of the hGR-S1(-349A) mRNA sequence resulted in a loss of the augmented response. The isoform hGR-S1(-349A) augments the response to steroids, and this significant response appears to be critically regulated by the 3'UTR. The identification and evaluation of these unique hGR isoforms helps further the understanding of the complex genetic regulation of the stress and steroid response.