To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4 degrees C inhibited the binding to hMR of both [3H]aldosterone and [3H]RU26752 (a synthetic aldosterone antagonist). At 4 degrees C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4 degrees C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20 degrees C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hmR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action.