The beta isoform of the human glucocorticoid receptor, hGRbeta, is a product of alternative splicing of the hGR gene. The physiological function of this isoform is unknown up to now. Recent data are contradictory in that they either favor or argue against a role of hGRbeta as a repressor of the functional hGRalpha isoform. In the present study hGRbeta did not inhibit transcriptional activation of the MMTV-driven luciferase reporter gene by dexamethasone-activated hGRalpha in COS-1 cells. In addition, two naturally occurring variants of the hGRbeta isoform associated with altered sensitivity to glucocorticoids, termed hGRbeta-R23K and hGRbeta-N363S, did not repress hGRalpha, even when overexpressed 10-fold. We conclude that the hGRbeta isoform, as well as two of its natural variants, do not act as dominant negative inhibitors of hGRalpha function and that the beta isoform does not appear to play a role in the regulation of glucocorticoid sensitivity.