Freshwater-adapted killifish (Fundulus heteroclitus) were transferred directly from soft fresh water to full-strength sea water for periods of 1 h, 3 h, 8 h and 1, 2, 7, 14 and 30 days. Controls were transferred to fresh water for 24 h. Measured variables included: blood [Na⁺], osmolality, glucose and cortisol levels, basal and stimulated rates of ion transport and permeability of in vitro opercular epithelium, gill Na⁺/K⁺-ATPase and citrate synthase activity and chloride cell ultrastructure. These data were compared with previously published killifish cystic fibrosis transmembrane conductance regulator (kfCFTR) expression in the gills measured over a similar time course. Plasma cortisol levels peaked at 1 h, coincident with a rise in plasma [Na⁺]. At 8 h after transfer to sea water, a time at which previous work has shown kfCFTR expression to be elevated, blood osmolality and [Na⁺] were high, and cortisol levels and opercular membrane short-circuit current (I_sc; a measure of Cl⁻ secretion rate) were low. The 24 h group, which showed the highest level of kfCFTR expression, had the highest plasma [Na⁺] and osmolality, elevated plasma cortisol levels, significantly lower opercular membrane resistance, an increased opercular membrane ion secretion rate and collapsed tubule inclusions in mitochondria-rich cells, but no change in gill Na⁺/K⁺-ATPase and citrate synthase activity or plasma glucose levels. Apparently, killifish have a rapid (<1 h) cortisol response to salinity coupled to subsequent (8–48 h) expression of kfCFTR anion channel proteins in existing mitochondria-rich cells that convert transport from ion uptake to ion secretion.