The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.