The common killifish, Fundulus heteroclitus, inhabits brackish water estuaries and salt marshes along the eastern coast of North America. The species distribution is latitudinal, from Newfoundland to Florida, and thus spans a cline of environmental temperatures. Correspondingly, many previous studies have investigated thermal adaptations in populations across the range. Differences between populations include latitudinal differences in glycolytic enzyme expression and activity (Powers et al., 1986;Pierce and Crawford, 1996), endocrinology (DeKoning et al., 2004;Picard and Schulte, 2004), metabolism (Podrabsky et al., 2000), morphology and behaviour (Powers et al., 1993), and, as a result, these fish are sometimes divided into two subspecies, F.h. macrolepidotus (northern) and F.h. heteroclitus (southern). By contrast, few studies have assessed whether intraspecific physiological differences exist between populations of F. heteroclitus in response to other environmental factors (e.g. tidal cycle; DiMichele and Westerman, 1997).Species within the genus Fundulus are suggested to have arisen from brackish water ancestors, and there is substantial variation in both the salinity of their native habitats (ranging from freshwater to seawater) and their salinity tolerance (Griffith, 1974). Intraspecific differences in salinity tolerance and distribution also appear to exist within some Fundulus species. For example, northern populations of F. heteroclitus have higher fertilization success and larval survival in hyposmotic salinities than southern populations (Able and Palmer, 1988). Furthermore, the proportion of northern genotypes increases in freshwater habitats, even at latitudes and temperatures that are typical for the southern subspecies (Powers et al., 1993). It is therefore likely that molecular or physiological differences exist within F. heteroclitus that form the basis for variation in freshwater tolerance. We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10·g·l -1 ) to freshwater. Differences in Na + regulation between each population might partially account for this difference in tolerance, because plasma Na + was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na + /K + -ATPase mRNA expression and activity in their gills to a greater extent 1-14·days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na + at 14·days. All observed differences in Na + regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl -regulation also existed between populations. Plasma Cl -was maintained in northern fish, but in southerns, plasma Cl -decrease...