The progesterone analog RU486, an abortifacient, inhibits the action of progestins in humans but not in chickens or hamsters. Substitution of cysteine at position 575 by glycine in the hormone binding domain (HBD) of the chicken progesterone receptor (cPR) generated a cPR that binds RU486 and whose activity is antagonized by that compound. In fact, all receptors that bind RU486 have a glycine at the corresponding position. The hamster PR, like cPR, has a cysteine. Only glycine--not methionine or leucine--at position 575 allowed binding of RU486 to cPR. Substitution of this glycine by cysteine in the human PR (hPR) abrogated binding of RU486 but not that of an agonist. The corresponding mutation in the human glucocorticoid receptor resulted in a loss of binding of both dexamethasone and RU486. Examination of a series of 11 beta-substituted steroids showed that antagonism is not an intrinsic property of an antihormone, because one hPR antagonist acted as an agonist for a mutated hPR. The positioning of an aromatic 11 beta-substitution in the PR HBD appears to be critical for generating agonistic or antagonistic activity.