Positive and negative selection steps in the thymus prevent non‐functional or harmful T cells from reaching the periphery. To examine the role of glucocorticoid (GC) hormone and its intracellular receptor (GCR) in thymocyte development we measured the GCR expression in different thymocyte subpopulations of BALB/c mice with or without previous dexamethasone (DX), anti‐CD3 mAb, RU‐486 and RU‐43044 treatment. Four‐color labeling of thymocytes allowed detection of surface CD4/CD8/CD69 expression in parallel with intracellular GCR molecules by flow cytometry. Double‐positive (DP) CD4⁺CD8⁺ thymocytes showed the lowest GCR expression compared to double‐negative (DN) CD4^–CD8^– thymocytes and mature single‐positive (SP) cells. DX treatment caused a concentration‐dependent depletion of the DP cell population and increased appearance of mature SP cells with reduced GCR levels. GCR antagonists (RU‐486 or RU‐43044) did not influence the effect of DX on thymocyte composition; however, RU‐43044 inhibited the high‐dose GC‐induced GCR down‐regulation in SP and DN cells. GCR antagonists alone did not influence the maturation of thymocytes and receptor numbers. Combined low‐dose anti‐CD3 mAb and DX treatment caused an enhanced maturation (positive selection) of thymocytes followed by the elevation of CD69⁺ DP cells. The sensitivity of DP thymocytes with a GCR^low phenotype to GC action and the ineffectiveness of the GCR antagonist treatment may reflect a non‐genomic GC action in the thymic selection steps.