The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group has previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone (DEX)-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that, even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR, and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein, hsp90. Co-incubation of the methoxyindole with DEX did not affect the expression of a reporter gene in GR transfected Cos-7 cells nor in HC11 and L929 mouse cell lines that express Mel-1A and ROR receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1A and ROR independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR mediated effects, which involves the inhibition of hsp90 dissociation and the cytoplasmic retention of the GR.