Glucocorticoids and mineralocorticoids have distinct in vivo roles despite close structural homology and similarities in vitro. Known mechanisms of specificity focus on factors extrinsic to the receptor; interactions that directly regulate the receptor to confer specificity are less well understood, particularly for the mineralocorticoid receptor (MR). To examine relative MR vs. glucocorticoid receptor (GR) function in a more physiological context, we compared transactivation by GR and MR in the standard experimental fibroblast CV-1 cell line, the renal epithelial LLC-PK1 line, and neuronal medullary raphe RN33B cells. Maximal transactivational activity mediated by MR, relative to that mediated by GR, is enhanced in both of these cell lines and is primarily conferred by an N-terminal-mediated enhancement of the MR response. In addition, the ligand concentration required for maximal transcriptional activity of the GR varies significantly between cell lines. This is independent of binding affinity or 11beta-hydroxysteroid dehydrogenase-mediated inactivation and may contribute to in vivo tissue-specific differences in responses to the GR. Although ligand binding affinity is clearly conferred by the LBD, receptor-specific variations between cell lines in transcriptional sensitivity to ligand appear, rather, to be associated with the N-terminus. These studies demonstrate that the specificity of the MR vs. the GR response may be mediated via unique cellular factors, as well as suggesting a novel means of expanding the cellular response to cortisol.