The role of corticosteroid receptors (CRs) in the regulation of gill permeability was examined using a primary cultured trout gill epithelium. The epithelium expressed both glucocorticoid receptors (GR1 and GR2) and a mineralocorticoid receptor (MR), and cortisol treatment significantly increased transepithelial resistance (TER) and decreased paracellular [(3)H]PEG-4000 flux. Epithelial permeability was unaffected by deoxycorticosterone or aldosterone. The GR antagonist RU486 as well as MR antagonists spironolactone and RU26752 significantly reduced, but did not completely block, the effects of cortisol. The MR antagonist eplerenone was without effect. Only RU486 + spironolactone or RU486 + RU26752 treatment completely suppressed the effects of cortisol. On its own, RU486 had cortisol-like effects which could be blocked by spironolactone, suggesting that although RU486 is a GR antagonist, in this system it may also have agonistic properties that are mediated through the MR. The GR agonist dexamethasone increased TER and reduced [(3)H]PEG-4000 flux across cultured epithelia and was unaffected by MR antagonists. Therefore, alterations in transcript abundance of select tight junction (TJ) proteins were examined in response to cortisol, dexamethasone (a GR agonist) and RU486 (as a MR agonist). Occludin and claudin-7, -8d, -12 and -31 mRNA were significantly elevated in response to cortisol, dexamethasone or RU486 treatment. Claudin-3a mRNA was significantly elevated in response to cortisol or dexamethasone only, and claudin-28b and -30 mRNA were significantly altered following cortisol or RU486 treatment only. The data support a role for the GRs and MR in regulating gill permeability and suggest that TJ proteins are responsive to cortisol through both or individual CR types.