The effect of 17β-estradiol (E 2) on osmoregulatory performance was examined in the euryhaline killifish, Fundulus heteroclitus. Fish were injected once with 1, 2 and 5 μg g −1 E 2 and, 6 h after injection, transferred from 1 ppt seawater (SW) to full strength SW (40 ppt) or from SW to 1 ppt SW. In another set of experiments, fish were injected four times on alternate days with 2 μg g −1 E 2 and then, 6 h after the last injection, transferred from 1 ppt SW to SW or from SW to 1 ppt SW. Fish were sampled 18 h after transfer (i.e., 24 h post-injection), and plasma osmolality, Na + and Cl − concentration and gill K +- pNPPase activity (a reflection of the sodium pump) were examined. Transfer from 1 ppt SW to SW resulted in significantly increased plasma osmolality, but did not affect gill K +- pNPPase activity. A single dose of E 2 (1, 2 and 5 μg g −1) prior to transfer from 1 ppt SW to SW increased plasma osmolality and decreased gill K +- pNPPase activity in a dose-dependent manner. Prolonged treatment with E 2 increased plasma osmolality and decreased gill K +- pNPPase activity in 1 ppt SW-adapted fish. Transfer of fish thus treated from 1 ppt SW to SW increased plasma osmolality and did not alter gill K +- pNPPase activity. Transfer from SW to 1 ppt SW had no significant effect on plasma osmolality or gill K +- pNPPase activity. Only the highest single dose of E 2 (5 μg g −1) prior to transfer from SW to 1 ppt SW decreased gill K +- pNPPase activity. Prolonged treatment with 2 μg g −1 E 2 decreased gill K +- pNPPase activity only following transfer from SW to 1 ppt SW. The results substantiate an inhibitory action of E 2 on hypoosmoregulatory capacity in this euryhaline teleost.