The rapid activation of gill Na⁺,K⁺-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25–35 ppt). In parr and presmolt, Salmo salar gill Na⁺,K⁺-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na⁺,K⁺-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na⁺,K⁺-ATPase, and the rapid increase after transfer to SW was not observed. Na⁺,K⁺-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na⁺,K⁺-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na⁺,K⁺-ATPase. Cycloheximide inhibited the increase in gill Na⁺,K⁺-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10^–4 M > 10^–5 M > 10^–6 M). Actinomycin D or bumetanide in the culture (doses of 10^–4 M, 10^–5 M, and 10^–6 M) did not affect gill Na⁺,K⁺-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na⁺,K⁺-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na⁺,K⁺-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes.