Background Congenital adrenal hyperplasia (CAH) patients are clinically often less severely affected by cortisol deficiency than anticipated from their enzymatic defect. We hypothesize that adrenal steroid hormone precursors that accumulate in untreated or poorly controlled CAH have glucocorticoid activity and partially compensate for cortisol deficiency. We studied the in vitro effects of 17-hydroxyprogesterone, progesterone, 21-deoxycortisol and androstenedione on the human glucocorticoid receptor (hGR). Methods Competitive binding assays were performed in HeLa cells. Nuclear translocation of the hGR was studied by transfection of COS-7 cells with a GFP-tagged hGR and fluorescence microscopy. Transactivation assays were performed in COS-7 cells and in HEK 293 cells after co-transfection with hGR and luciferase reporter vectors using a dual luciferase assay. Results 17-hydroxyprogesterone, progesterone and 21-deoxycortisol are able to bind to the hGR with binding affinities of 24 - 43% compared to cortisol. Androstenedione has a low binding affinity. Incubation with 21-deoxycortisol led to complete nuclear translocation of the hGR, whereas treatment with 17-hydroxyprogesterone or progesterone resulted in partial nuclear translocation. 21-deoxycortisol transactivated the hGR with an EC50 approximately 6-fold the EC50 of cortisol. 17-hydroxyprogesterone and progesterone transactivated the hGR with EC50s of more than 100 times the EC50 of cortisol. No hGR transactivation was detected after incubation with androstenedione. Conclusions 21-deoxycortisol, 17-hydroxyprogesterone and progesterone are able to bind, translocate and transactivate the hGR in vitro and thus may have glucocorticoid activity. Mainly 21-deoxycortisol might have a clinically relevant agonistic effect on the hGR and could potentially partially compensate the cortisol deficiency in CAH patients.