Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na+-K+-ATPase activity of Atlantic salmon smolts. A major α-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The α-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in α-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na+-K+-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in α-mRNA abundance and had basal levels of α-protein and enzyme activity. Measurement of the binding of [3H]ouabain to Na+-K+-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20–23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in α-mRNA levels after 24 h with a rise in Na+-K+-ATPase activity occurring only after 11 days. No qualitative change in α-expression was revealed at either the mRNA or protein level. Immunological identification of the α-protein was performed with polyclonal antibodies directed against the rat α-specific isoforms, revealing that parr, freshwater, and seawater smolts have an α3-like isoform. This study shows that the increase in Na+-K+-ATPase activity in smolt gills depends first on an increase in the α-mRNA expression and is followed by a slower rise in α-protein abundance that eventually leads to a higher synthesis of Na+-K+pumps.