Previous attempts to characterize the structural determinants required for binding of cortisol by the glucocorticoid receptor (GR) have proved difficult since almost all modifications of the ligand binding domain (LBD) of GRs either eliminate or greatly decrease steroid binding. The guinea pig, a New World hystricomorph with a phylogeny the subject of recent dispute, is corticoresistant due to a GR that has diminished affinity for dexamethasone. The guinea pig GR has been cloned, and sequencing has identified many unique amino acid substitutions in the LBD. Using a domain-swap approach, the cloned guinea pig GR LBD was substituted for the human GR LBD in a human GR expression vector. Dexamethasone response curves for these constructs show that the cortisol resistance observed in the guinea pig in vivo is conferred in vitro by the guinea pig GR LBD. In addition, the guinea pig GR LBD induces a high level of constitutive activity. This constitutive activity is not repressed by RU486 (1 μM) but is enhanced by the addition of 8-bromo-cAMP. One of the amino acid substitutions results in the loss of a cysteine, which in the human, rat, and mouse GR is the site of covalent attachment for dexamethasone-21-mesylate. This cysteine is replaced by a tryptophan residue in the guinea pig GR, the implications of which were examined by reciprocal mutation of the tryptophan to a cysteine in the guinea pig GR LBD, and the cystine to a tryptophan in the human GR LBD. The human GR mutant receptor retains wild-type activity; however, mutation of tryptophan to a cysteine in the guinea pig GR LBD increases dexamethasone sensitivity by 1 order of magnitude and abolishes the constitutive activity. The tryptophan is therefore a key residue necessary for both decreased glucocorticoid sensitivity and the unexpected constitutive activity of the guinea pig GR. The guinea pig thus provides a naturally 'altered' yet functional GR in which the structural determinants of GR-ligand binding as well as the molecular basis of cortisol resistance may be studied.