Few studies have examined changes in salmon gill ion transporter expression during the transition from seawater to freshwater, a pivotal moment in the salmonid life cycle. Seawater-acclimated Atlantic salmon were transferred to freshwater and blood and gill tissue were sampled over 30 days of acclimation. Salmon held in seawater had stable plasma osmolality and sodium and chloride levels throughout the experiment. Following freshwater exposure, plasma sodium and chloride levels and total osmolality decreased significantly before returning towards control levels over time. Gill H(+)-ATPase activity increased by more than 45% 14 days after exposure to freshwater, whereas H(+)-ATPase mRNA levels were not affected by the salinity change. Within 4 days of freshwater exposure, gill Na(+)/K(+)-ATPase activity increased ∼43% over control levels, remaining significantly higher until the 30 day sampling group when it declined back to control levels. This increase in activity was associated with a more than 7-fold increase in Na(+)/K(+)-ATPase isoform α1a mRNA level and a ∼60% decrease in Na(+)/K(+)-ATPase isoform β1b mRNA level. The mRNA levels of Na(+)/K(+)-ATPase isoforms α1c and α3 did not change as a result of freshwater exposure. The time courses for mRNA expression of the small membrane protein FXYD 11 and the β1-subunit were very similar, with levels increasing significantly 7 days following freshwater exposure before subsiding back to control levels at 30 days. Taken together, these data suggest an important role for Na(+)/K(+)-ATPase in freshwater acclimation in Atlantic salmon.