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For the current European legislation, the chemical analysis of drug residues is the exclusive accepted method to identify animals illicitly treated with growth promoters. Glucocorticoids and their metabolites are no detectable by LC/MS-MS methods in biological fluids when the growth promoter administration is discontinued several days prior to the slaughtering. The aim of this study was to elucidate the effect on the expression of genes belonging to the glucocorticoid pathway in three types of skeletal muscle of calves treated with prednisolone or dexamethasone in combination with estradiol. A gene expression change of glucocorticoid receptors (NR3C1 and NR3C2), their chaperones molecules (FKBP prolyl isomerase 4 and 5, FKBP4 and 5) and pre-receptor system (hydroxysteroid 11-beta dehydrogenases 1 and 2, HSD11B1 and 2) may indicate potential biomarkers of glucocorticoid treatment. In the biceps brachii muscle, the administration of dexamethasone with estradiol increased HSD11B2 (P 0.01) and NR3C2 (P 0.01) gene expression, whereas prednisolone administration increased HSD11B1 transcript levels (P 0.05). In the longissimus lumborum muscle, NR3C2 gene expression decreased following prednisolone administration (P 0.05). FKBP5 gene expression decreased in all considered muscles of calves administered with dexamethasone and estradiol (P 0.01), whereas increased in the longissimus lumborum (P 0.01) and vastus lateralis (P 0.05) muscle of prednisolone-treated group (P 0.05). The opposite effect of dexamethasone and prednisolone appears very promising to develop a low-cost screening test, because the expression analysis of a unique gene in a given tissue may distinguish the dispensed molecules.