To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10???) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg-1) or high dose (200 mg.kg-1). Fish were transferred to 1.5 x seawater (45???) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors. ?? 2005 Wiley-Liss, Inc.