AbstractDuring the colonization of freshwater by marine fish, adaptation to hypoosmotic conditions may impact their ability to osmoregulate in seawater. The prickly sculpin (Cottus asper) is a euryhaline fish with marine ancestors that postglacially colonized many freshwater habitats. Previous work on C. asper suggested that isolation in freshwater habitats has resulted in putative adaptations that improve ion regulation in freshwater populations compared with populations with current access to estuaries. To determine whether long-term colonization of freshwater is associated with a reduced ability to ion regulate in seawater, we acclimated C. asper populations from three habitat types that vary in the extent to which they are isolated from marine habitats and compared their seawater osmoregulation. Seawater acclimation revealed that lake populations exhibited a reduced capacity to osmoregulate in seawater compared with coastal river populations with ongoing access to estuaries. In particular, when acclimated to seawater for several weeks, lake populations had lower gill Na+/K+-ATPase activity and lower intestinal H+-ATPase activity than coastal river populations. Lake populations also had a reduced ability to maintain plasma ion concentrations, and they produced lower quantities of intestinal carbonate precipitates in seawater than coastal river populations. Furthermore, there was a positive relationship between the anterior intestinal Na+/K+-ATPase activity and the amount of precipitate produced by the intestine, which suggests that the anterior intestine plays a role in seawater osmoregulation. Our results suggest that the extent of isolation from the sea could, in part, explain the reduced osmoregulation in seawater in postglacial freshwater populations of C. asper.