Steroid hormone receptor antagonists are widely used in clinical medicine, but their use is often complicated by the lack of receptor specificity to presently available drugs. We previously demonstrated an important role of a widely conserved helix 3 (H3)-helix 5 (H5) interaction in determining the sensitivity and specificity of steroid hormone receptors to receptor agonists. Intriguingly, the same H3 residues also play a crucial role in receptor antagonism; mutation of these residues alters the response of these receptors to antagonists. Given the close interaction of H3 and H5 residues at this site, we asked whether H5 residues might also play a role in the sensitivity of these receptors to antagonists. We demonstrate here that modification of H5 residues produces marked changes in the sensitivities of the glucocorticoid and progesterone receptor (PR) to RU486 antagonism. Moreover, while we confirm previous reports that alteration of the H3 residue, Gly 722 prevents RU486-mediated inhibition of the PR, we show that the corresponding substitution in the glucocorticoid receptor does not inhibit RU486-mediated receptor antagonism. Taken together, our data support the notion that RU486 binds differently to these two receptors, providing a potential target for the design of more specific antiglucocorticoid and antiprogestin drugs.