The proposed mineralocorticoid-like signalling axis in teleost fish, consisting of the hormone 11-deoxycorticosterone (DOC) and a mineralocorticoid receptor (MR), has recently challenged our conception of cortisol being the only osmoregulatory corticosteroid in teleost fish. This paper aimed at comparing the osmoregulatory role of DOC with that of cortisol during the pre-adaptive development of SW-tolerance, smoltification, in Atlantic salmon. Using an in vitro gill block incubation system, the effect of DOC and cortisol in the gill was investigated from January to September, using Na(+),K(+)-ATPase alpha-subunit isoforms alpha-1a and alpha-1b mRNA levels as targets for regulation by the hormones. Cortisol and DOC both conferred significant up-regulation of alpha-1a and alpha-1b mRNA levels at specific time-points during smoltification. However, the effect of cortisol and DOC on alpha-subunit isoforms varied seasonally between isoforms and hormones. The maximum induction of alpha-1a was 3- to 4-fold compared to controls whereas a 2-fold induction was observed for alpha-1b. The pattern and capacity of stimulation of alpha-1a through smoltification were similar for cortisol and DOC, whereas cortisol had an enhanced capacity to stimulate alpha-1b as compared to DOC. Even though there was no demonstrable change in cortisol or DOC sensitivity in the gill, the magnitude of the hormonal effects were seasonally dependent. This is the first report of DOC-induced effects on osmoregulatory targets in fish, thus indicating a role for DOC and MR signalling in osmoregulation.